Antimicrobial/adjuvant compounds and methods

ABSTRACT

Among other things, in general, antimicrobial and/or adjuvant compounds are provided according to Formula Ia: (Ia) in which E and R1-11 have the meanings described herein; and prodrugs and pharmaceutically acceptable salts thereof. Other formulae and methods of use are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.No. 61/448,682, filed Mar. 3, 2011, the content of which is herebyincorporated by reference in its entirety.

BACKGROUND

Infection is an invasion of a host organism by a foreign organism,generally to the detriment of normal function in the host. In treatinghumans and other animals for infection and post-infective inflammatorydisease (e.g. septic shock), practitioners usually rely on chemicalcompounds known to have antibiotic affects, whether antiviral,antibacterial, antifungal, or the like.

Unfortunately, many pathogens have become resistant to currentantibiotic treatments. Antibiotic resistance is therefore anincreasingly significant clinical issue, calling for novel antibiotics.Especially valuable would be new antimicrobial adjuvant compounds,which, while not necessarily antimicrobial themselves, can increase thepotency, efficacy, and/or spectrum of activity of antibiotics whenco-administered or given as combination therapy. There is a need for newantimicrobials and new antimicrobial adjuvant compounds.

SUMMARY

The compounds of the present invention are of utility in antimicrobialinfections. In some cases, the compounds are themselves antimicrobial.In some cases, the compounds (“antimicrobial adjuvants”) have beneficialeffects in conjunction with an antimicrobial, reducing the dose ofantibiotic required for antimicrobial activity when administered incombination. In some cases, the compounds are both antimicrobial andantimicrobial adjuvants.

In general, in an aspect, compounds of Formula Ia are provided:

in which E is —CH₂— or is absent such that thiophene is directlyconnected to phenyl; R₁, R₂, R₉, R₁₀, and R₁₁ are each independentlyhydrogen, methyl, ethyl, propyl, cyclopropyl, butyl, cyclobutyl, pentyl,hexyl, isopropryl, isobutyl, neopentyl, methoxy, or ethoxy;additionally, R₁ and R₂ may connect to form a phenyl or benzofuran ring;additionally, R₉ and R₁₀ may connect to form a phenyl or benzofuranring; R₃ and R₈ are each independently hydrogen, methyl, ethyl, propyl,isopropyl, chlorine, fluorine, tert-butyl, methylsulfonyl, methoxy, orethoxy; R₄ and R₇ are each independently hydrogen, chlorine, methylester, ethyl ester, methyl, ethyl, propyl, cyclopropyl, butyl,cyclobutyl, isopropryl, isobutyl, methoxy, or ethoxy; and R₅ and R₆ areeach independently hydrogen, cyclopentyl, cyclopropyl, furan, thiophene,trifluoromethyl, trifluoromethyl ether, methylthiol, formaldehyde,chlorine, fluorine, bromine, phenyl, methyl, ethyl, isopropyl, propyl,butyl, cyclobutyl, isobutyl, neopentyl, pentyl, methoxy, or ethoxy.

In general, in an aspect of the invention, a method of treatment of amicrobial infection is provided including administering an effectiveamount of an antimicrobial compound disclosed herein to a patient inneed thereof.

In general, in an aspect of the invention, a method of treatment of amicrobial infection is provided including administering an effectiveamount of an antimicrobial adjuvant compound disclosed herein and aneffective amount of an antimicrobial compound to a patient in needthereof.

Compounds of other formulae are provided as described in the DetailedDescription below.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts thiophene- or thiazole-core compounds and associatedcompound identifiers of the present invention, along with theirrespective minimum inhibitory concentrations against MRSA generallyfollowing the procedure in Example 9 (“MRSA_MIC” reported in μM).

FIG. 2 depicts phenyl-core compounds and associated compound identifiersof the present invention.

DETAILED DESCRIPTION Definitions

Unless otherwise defined, terms as used in the specification refer tothe following definitions, as detailed below.

The term “acyl” as used herein means an alkyl group, as defined herein,appended to the parent molecular moiety through a carbonyl group, asdefined herein. Representative examples of acyl include, but are notlimited to, acetyl, 1-oxopropyl, 2,2-dimethyl-1-oxopropyl, 1-oxobutyl,and 1-oxopentyl.

The term “acyloxy” as used herein means an acyl group, as definedherein, appended to the parent molecular moiety through an oxygen atom.Representative examples of acyloxy include, but are not limited to,acetyloxy, propionyloxy, and isobutyryloxy.

The terms “administration” or “administering” compound should beunderstood to mean providing a compound of the present invention to anindividual in a form that can be introduced into that individual's bodyin an amount effective for prophylaxis, treatment, or diagnosis, asapplicable. Such forms may include for example oral dosage forms,injectable dosage forms, transdermal dosage forms, inhalation dosageforms, and rectal dosage forms.

The term “alkenyl” as used herein means a straight chain, branchedand/or cyclic hydrocarbon having from 2 to 20 (e.g., 2 to 10 or 2 to 6)carbon atoms, and including at least one carbon-carbon double bond.Representative alkenyl moieties include vinyl, allyl, 1-butenyl,2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl,2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, 1-hexenyl, 2-hexenyl,3-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 1-octenyl, 2-octenyl,3-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 2-decenyl and3-decenyl.

The term “alkoxy” as used herein means an alkyl group, as definedherein, appended to the parent molecular moiety through an oxygen atom.Representative examples of alkoxy include, but are not limited to,methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, andhexyloxy.

The term “alkoxyalkoxy” as used herein means an alkoxy group, as definedherein, appended to the parent molecular moiety through another alkoxygroup, as defined herein. Representative examples of alkoxyalkoxyinclude, but are not limited to, tert-butoxymethoxy, 2-ethoxyethoxy,2-methoxyethoxy, and methoxymethoxy.

The term “alkoxyalkyl” as used herein means an alkoxy group, as definedherein, appended to the parent molecular moiety through an alkyl group,as defined herein. Representative examples of alkoxyalkyl include, butare not limited to, tert-butoxymethyl, 2-ethoxyethyl, 2-methoxyethyl,and methoxymethyl.

The term “alkoxycarbonyl” as used herein means an alkoxy group, asdefined herein, appended to the parent molecular moiety through acarbonyl group, as defined herein. Representative examples ofalkoxycarbonyl include, but are not limited to, methoxycarbonyl,ethoxycarbonyl, and tert-butoxycarbonyl.

The term “alkoxyimino” as used herein means an alkoxy group, as definedherein, appended to the parent molecular moiety through an imino group,as defined herein. Representative examples of alkoxyimino include, butare not limited to, ethoxy(imino)methyl and methoxy(imino)methyl.

The term “alkoxysulfonyl” as used herein means an alkoxy group, asdefined herein, appended to the parent molecular moiety through asulfonyl group, as defined herein. Representative examples ofalkoxysulfonyl include, but are not limited to, methoxysulfonyl,ethoxysulfonyl, and propoxysulfonyl.

The term “alkyl” as used herein means a straight or branched chainhydrocarbon containing from 1 to 20 carbon atoms, preferably from 1 to10 carbon atoms, more preferably 1, 2, 3, 4, 5, or 6 carbons.Representative examples of alkyl include, but are not limited to,methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl,tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl,2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, andn-decyl.

The term “alkylamino” as used herein means an alkyl group, as definedherein, appended to the parent molecular moiety through a NH group.Representative examples of alkylamino include, but are not limited to,methylamino, ethylamino, isopropylamino, and butylamino.

The term “alkylcarbonyl” as used herein means an alkyl group, as definedherein, appended to the parent molecular moiety through a carbonylgroup, as defined herein. Representative examples of alkylcarbonylinclude, but are not limited to, methylcarbonyl, ethylcarbonyl,isopropylcarbonyl, n-propylcarbonyl, and the like.

The term “alkylsulfonyl” as used herein means an alkyl group, as definedherein, appended to the parent molecular moiety through a sulfonylgroup, as defined herein. Representative examples of alkylsulfonylinclude, but are not limited to, methylsulfonyl and ethylsulfonyl.

The term “alkynyl” as used herein means a straight or branched chainhydrocarbon group containing from 2 to 10 carbon atoms, and preferably2, 3, 4, or 5 carbons, and containing at least one carbon-carbon triplebond. Representative examples of alkynyl include, but are not limitedto, acetylenyl, 1-propynyl, 2-propynyl, 3-butynyl, 2-pentynyl, and1-butynyl.

The term “amido” as used herein means an amino, alkylamino, ordialkylamino group appended to the parent molecular moiety through acarbonyl group, as defined herein. Representative examples of amidoinclude, but are not limited to, aminocarbonyl, methylaminocarbonyl,dimethylaminocarbonyl, and ethylmethylaminocarbonyl.

The term “amino” as used herein means a —NH₂ group.

The term “aryl” as used herein means a monocyclic hydrocarbon aromaticring system. Representative examples of aryl include, but are notlimited to, phenyl.

The term “arylalkyl” as used herein means an aryl group, as definedherein, appended to the parent molecular moiety through an alkyl group,as defined herein. Representative examples of arylalkyl include, but arenot limited to, benzyl, 2-phenylethyl and 3-phenylpropyl.

The term “carbonyl” as used herein means a —C(═O)— group.

The term “carboxy” as used herein means a —COOH group, which may beprotected as an ester group: —COO-alkyl.

The term “cyano” as used herein means a —CN group.

The term “cyanophenyl” as used herein means a —CN group appended to theparent molecular moiety through a phenyl group, including, but notlimited to, 4-cyanophenyl, 3-cyanophenyl, and 2-cyanophenyl.

The term “cycloalkyl” as used herein means a saturated cyclichydrocarbon group containing from 3 to 8 carbons. Examples of cycloalkylinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,and cyclooctyl.

The term “cycloalkylcarbonyl” as used herein means a cycloalkyl group,as defined herein, appended to the parent molecular moiety through acarbonyl group, as defined herein. Representative examples ofcycloalkylcarbonyl include, but are not limited to, cyclopropylcarbonyl,cyclopentylcarbonyl, cyclohexylcarbonyl, and cycloheptylcarbonyl.

The term “dialkylamino” as used herein means two independent alkylgroups, as defined herein, appended to the parent molecular moietythrough a nitrogen atom. Representative examples of dialkylaminoinclude, but are not limited to, dimethylamino, diethylamino,ethylmethylamino, and butylmethylamino.

The term “fluoro” as used herein means —F.

The term “fluoroalkoxy” as used herein means at least one fluoroalkylgroup, as defined herein, appended to the parent molecular moietythrough an oxygen group, as defined herein. Representative examples offluoroalkyl include, but are not limited to, trifluoromethoxy (CF₃O—),and difluoromethoxy (CHF₂O—).

The term “fluoroalkyl” as used herein means at least one fluoro group,as defined herein, appended to the parent molecular moiety through analkyl group, as defined herein. Representative examples of fluoroalkylinclude, but are not limited to, fluoromethyl, difluoromethyl,trifluoromethyl, pentafluoroethyl, and 2,2,2-trifluoroethyl.

The term “formyl” as used herein means a —C(O)H group.

The term “halo” or “halogen” as used herein means Cl, Br, I, or F.

The term “haloalkoxy” as used herein means at least one halogen, asdefined herein, appended to the parent molecular moiety through analkoxy group, as defined herein. Representative examples of haloalkoxyinclude, but are not limited to, 2-fluoroethoxy, trifluoromethoxy, andpentafluoroethoxy.

The term “haloalkyl” as used herein means at least one halogen, asdefined herein, appended to the parent molecular moiety through an alkylgroup, as defined herein. Representative examples of haloalkyl include,but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl,pentafluoroethyl, and 2-chloro-3-fluoropentyl.

The term “heteroaryl”, as used herein, refers to an aromatic ringcontaining one or more heteroatoms independently selected from nitrogen,oxygen, or sulfur, or a tautomer thereof. Such rings can be monocyclicor bicyclic as further described herein. Heteroaryl rings are connectedto the parent molecular moiety through a carbon or nitrogen atom.

The terms “monocyclic heteroaryl” or “5- or 6-membered heteroaryl ring”,as used herein, refer to 5- or 6-membered aromatic rings containing 1,2, 3, or 4 heteroatoms independently selected from nitrogen, oxygen, orsulfur, or a tautomer thereof. Examples of such rings include, but arenot limited to, a ring wherein one carbon is replaced with an O or atom;one, two, or three N atoms arranged in a suitable manner to provide anaromatic ring; or a ring wherein two carbon atoms in the ring arereplaced with one O or S atom and one N atom. Such rings can include,but are not limited to, a six-membered aromatic ring wherein one to fourof the ring carbon atoms are replaced by nitrogen atoms, five-memberedrings containing a sulfur, oxygen, or nitrogen in the ring; fivemembered rings containing one to four nitrogen atoms; and five memberedrings containing an oxygen or sulfur and one to three nitrogen atoms.Representative examples of 5- to 6-membered heteroaryl rings include,but are not limited to, furyl, imidazolyl, isoxazolyl, isothiazolyl,oxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridinyl, pyrimidinyl,pyrrolyl, tetrazolyl, [1,2,3]thiadiazolyl, [1,2,3]oxadiazolyl,thiazolyl, thienyl, [1,2,3]triazinyl, [1,2,4]triazinyl,[1,3,5]triazinyl, [1,2,3]triazolyl, and [1,2,4]triazolyl. The term“bicyclic heteroaryl” or “8- to 12-membered bicyclic heteroaryl ring”,as used herein, refers to an 8-, 9-, 10-, 11-, or 12-membered bicyclicaromatic ring containing at least 3 double bonds, and wherein the atomsof the ring include one or more heteroatoms independently selected fromoxygen, sulfur, and nitrogen. Representative examples of bicyclicheteroaryl rings include indolyl, benzothienyl, benzofuranyl, indazolyl,benzimidazolyl, benzothiazolyl, benzoxazolyl, benzoisothiazolyl,benzoisoxazolyl, quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinyl,phthalazinyl, pteridinyl, purinyl, naphthyridinyl, cinnolinyl,thieno[2,3-d]imidazole, thieno[3,2-b]pyridinyl, and pyrrolopyrimidinyl.

The terms “heterocyclic ring” and “heterocycle”, as used herein, referto a 4- to 12-membered monocyclic or bicyclic ring containing one, two,three, four, or five heteroatoms independently selected from the groupconsisting of nitrogen, oxygen, and sulfur and also containing either atleast one carbon atom attached to four other atoms or one carbon atomsubstituted with an oxo group and attached to two other atoms. Four- andfive-membered rings may have zero or one double bond. Six-membered ringsmay have zero, one, or two double bonds. Seven- and eight-membered ringsmay have zero, one, two, or three double bonds. The non-aromaticheterocycle groups of the invention can be attached through a carbonatom or a nitrogen atom. The non-aromatic heterocycle groups may bepresent in tautomeric form. Representative examples ofnitrogen-containing heterocycles include, but are not limited to,azepanyl, azetidinyl, aziridinyl, azocanyl, dihydropyridazinyl,dihydropyridinyl, dihydropyrimidinyl, morpholinyl, piperazinyl,piperidinyl, pyrrolidinyl, pyrrolinyl, dihydrothiazolyl,dihydropyridinyl, and thiomorpholinyl. Representative examples ofnon-nitrogen containing non-aromatic heterocycles include, but are notlimited to, dioxanyl, dithianyl, tetrahydrofuryl, dihydropyranyl,tetrahydropyranyl, and [1,3]dioxolanyl.

The term “hydroxy” as used herein means an —OH group.

The term “hydroxyalkyl” as used herein means at least one hydroxy group,as defined herein, appended to the parent molecular moiety through analkyl group, as defined herein. Representative examples of hydroxyalkylinclude, but are not limited to, hydroxymethyl, 2-hydroxyethyl,2-methyl-2-hydroxyethyl, 3-hydroxypropyl, 2,3-dihydroxypentyl, and2-ethyl-4-hydroxyheptyl.

The term “hydroxy-protecting group” means a substituent which protectshydroxyl groups against undesirable reactions during syntheticprocedures. Examples of hydroxy-protecting groups include, but are notlimited to, methoxymethyl, benzyloxymethyl, 2-methoxyethoxymethyl,2-(trimethylsilyl)ethoxymethyl, benzyl, triphenylmethyl,2,2,2-trichloroethyl, t-butyl, trimethylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, methylene acetal, acetonide benzylidene acetal,cyclic ortho esters, methoxymethylene, cyclic carbonates, and cyclicboronates. Hydroxy-protecting groups are appended onto hydroxy groups byreaction of the compound that contains the hydroxy group with a base,such as triethylamine, and a reagent selected from an alkyl halide,alkyl trifilate, trialkylsilyl halide, trialkylsilyl triflate,aryldialkylsilyltriflate, or an alkylchloroformate, CH₂I₂, or adihaloboronate ester, for example with methyliodide, benzyl iodide,triethylsilyltriflate, acetyl chloride, benzylchloride, ordimethylcarbonate. A protecting group also may be appended onto ahydroxy group by reaction of the compound that contains the hydroxygroup with acid and an alkyl acetal.

The term “imino” as defined herein means a —C(═NH)— group.

The term “mercapto” as used herein means a —SH group.

The term “nitro” as used herein means a —NO₂ group.

The term “nitrogen protecting group” as used herein means those groupsintended to protect a nitrogen atom against undesirable reactions duringsynthetic procedures. Nitrogen protecting groups comprise carbamates,amides, N-benzyl derivatives, and imine derivatives. Preferred nitrogenprotecting groups are acetyl, benzoyl, benzyl, benzyloxycarbonyl (Cbz),formyl, phenylsulfonyl, pivaloyl, tert-butoxycarbonyl (Boc),tert-butylacetyl, trifluoroacetyl, and triphenylmethyl (trityl).Nitrogen-protecting groups are appended onto primary or secondary aminogroups by reacting the compound that contains the amine group with base,such as triethylamine, and a reagent selected from an alkyl halide, analkyl trifilate, a dialkyl anhydride, for example as represented by(alkyl-O) 2 C═O, a diaryl anhydride, for example as represented by(aryl-O) 2 C═O, an acyl halide, an alkylchloroformate, or analkylsulfonylhalide, an arylsulfonylhalide, or halo-CON(alkyl)2, forexample acetylchloride, benzoylchloride, benzylbromide,benzyloxycarbonylchloride, formylfluoride, phenylsulfonylchloride,pivaloylchloride, (tert-butyl-O—C═O) 2 O, trifluoroacetic anhydride, andtriphenylmethylchloride.

The term “oxo” as used herein means (═O).

Unless otherwise indicated, the term “prodrug” encompassespharmaceutically acceptable esters, carbonates, thiocarbonates, N-acylderivatives, N-acyloxyalkyl derivatives, quaternary derivatives oftertiary amines, N-Mannich bases, Schiff bases, amino acid conjugates,phosphate esters, metal salts and sulfonate esters of compoundsdisclosed herein. Examples of prodrugs include compounds that comprise abiohydrolyzable moiety (e.g., a biohydrolyzable amide, biohydrolyzablecarbamate, biohydrolyzable carbonate, biohydrolyzable ester,biohydrolyzable phosphate, or biohydrolyzable ureide analog). Prodrugsof compounds disclosed herein are readily envisioned and prepared bythose of ordinary skill in the art. See, e.g., Design of Prodrugs,Bundgaard, A. Ed., Elseview, 1985; Bundgaard, hours, “Design andApplication of Prodrugs,” A Textbook of Drug Design and Development,Krosgaard-Larsen and hours. Bundgaard, Ed., 1991, Chapter 5, p. 113-191;and Bundgaard, hours, Advanced Drug Delivery Review, 1992, 8, 1-38.

Unless otherwise indicated, the term “protecting group” or “protectivegroup,” when used to refer to part of a molecule subjected to a chemicalreaction, means a chemical moiety that is not reactive under theconditions of that chemical reaction, and which may be removed toprovide a moiety that is reactive under those conditions. Protectinggroups are well known in the art. See, e.g., Greene, T. W. and Wuts, P.G. M., Protective Groups in Organic Synthesis (3 rd ed., John Wiley &Sons: 1999); Larock, R. C., Comprehensive Organic Transformations (2nded., John Wiley & Sons: 1999). Some examples include benzyl,diphenylmethyl, trityl, Cbz, Boc, Fmoc, methoxycarbonyl, ethoxycarbonyl,and phthalimido. Protecting groups include, for example, nitrogenprotecting groups and hydroxy-protecting groups.

The term “sulfonyl” as used herein means a —S(O)₂— group.

The term “thioalkoxy” as used herein means an alkyl group, as definedherein, appended to the parent molecular moiety through a sulfur atom.Representative examples of thioalkoxy include, but are no limited to,methylthio, ethylthio, and propylthio.

Certain compounds of the present invention may exist as stereoisomerswherein, asymmetric or chiral centers are present. These stereoisomersare “R” or “S” depending on the configuration of substituents around thechiral carbon atom. The terms “R” and “S” used herein are configurationsas defined in IUPAC 1974 Recommendations for Section E, FundamentalStereochemistry, in Pure Appl. Chem., 1976, 45: 13-30. The inventioncontemplates various stereoisomers and mixtures thereof and these arespecifically included within the scope of this invention. Stereoisomersinclude enantiomers and diastereomers, and mixtures of enantiomers ordiastereomers. Individual stereoisomers of compounds of the inventionmay be prepared synthetically from commercially available startingmaterials which contain asymmetric or chiral centers or by preparationof racemic mixtures followed by resolution well known to those ofordinary skill in the art. These methods of resolution are exemplifiedby (1) attachment of a mixture of enantiomers to a chiral auxiliary,separation of the resulting mixture of diastereomers byrecrystallization or chromatography and optional liberation of theoptically pure product from the auxiliary as described in Furniss,Hannaford, Smith, and Tatchell, “Vogel's Textbook of Practical OrganicChemistry”, 5th edition (1989), Longman Scientific & Technical, EssexCM20 2JE, England, incorporated herein by reference for the disclosureof methods for separation and purification of diastereomers or (2)direct separation of the mixture of optical enantiomers on chiralchromatographic columns or (3) fractional recrystallization methods.

Certain compounds of the present invention may exist as cis or transisomers, wherein substituents on a ring may attached in such a mannerthat they are on the same side of the ring-(cis) relative to each other,or on opposite sides of the ring relative to each other (trans). Suchmethods are well known to those of ordinary skill in the art, and mayinclude separation of isomers by recrystallization or chromatography. Itshould be understood that the compounds of the invention may possesstautomeric forms, as well as geometric isomers, and that these alsoconstitute an aspect of the invention.

It should be noted that a chemical moiety that forms part of a largercompound may be described herein using a name commonly accorded it whenit exists as a single molecule or a name commonly accorded its radical.For example, the terms “pyridine” and “pyridyl” are accorded the samemeaning when used to describe a moiety attached to other chemicalmoieties. Thus, for example, the two phrases “XOH, wherein X is pyridyl”and “XOH, wherein X is pyridine” are accorded the same meaning, andencompass the compounds pyridin-2-ol, pyridin-3-ol and pyridin-4-ol.

It should also be noted that names of compounds having one or morechiral centers that do not specify the stereochemistry of those centersencompass pure stereoisomers and mixtures thereof. Moreover, any atomshown in a drawing with unsatisfied valences is assumed to be attachedto enough hydrogen atoms to satisfy the valences. In addition, chemicalbonds depicted with one solid line parallel to one dashed line encompassboth single and double (e.g., aromatic) bonds, if valences permit.

The term “pharmaceutically acceptable excipient”, as used herein, meansa non-toxic, inert solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type. Someexamples of materials which can serve as pharmaceutically acceptablecarriers are sugars such as lactose, glucose and sucrose; starches suchas corn starch and potato starch; cellulose and its derivatives such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; cocoa butter and suppositorywaxes; oils such as peanut oil, cottonseed oil, safflower oil, sesameoil, olive oil, corn oil and soybean oil; glycols; such a propyleneglycol; esters such as ethyl oleate and ethyl laurate; agar; bufferingagents such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol,and phosphate buffer solutions, as well as other non-toxic compatiblelubricants such as sodium lauryl sulfate and magnesium stearate, as wellas coloring agents, releasing agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the composition, according to the judgment of one skilledin the art of formulations.

Unless otherwise indicated, a “therapeutically effective amount” of acompound is an amount sufficient to treat a disease or condition, or oneor more symptoms associated with the disease or condition. In someembodiments, “treatment” may be determined by comparison to an untreatedcontrol.

The term “subject” is intended to include living organisms in whichdisease may occur. Examples of subjects include humans, monkeys, cows,sheep, goats, dogs, cats, mice, rats, and transgenic species thereof.

The present invention is based, at least in part, on the design ofcompounds that inhibit acyl carrier protein (ACP) synthase (AcpS), theenzyme responsible for converting apo-ACP into holo-ACP. AcpS is presentnot only in Gram-positive and -negative bacteria, but also in acid-fastbacteria such as Mycobacterium tuberculosis, and even in protozoa suchas Plasmodium falciparum. Accordingly, AcpS inhibitors are expected tohave deleterious effects on the viability of many microbes. AcpSinhibitors are be expected to have deleterious effects on themaintenance of microbial cell function, including, for example,increased porosity of cell membranes and dysfunction of efflux pumps insuch membranes. The spectrum of activity demonstrated for compounds ofthe present invention includes many Gram-positive bacteria includingMethicillin-resistant S. aureus, with selected compounds retainingactivity against Gram-negative pathogens such as Ps. aeruginosa, Ac.baumanii, and S. maltophilia, and the like. Compounds of the presentinvention with low or no antimicrobial potency against, for example, Ps.aeruginosa, may have antimicrobial adjuvant (“adjuvant” or“pro-antibiotic”) effects when co-administered with antimicrobialcompounds such as, for example, azithromycin, erythromycin, orampicillin, or with antimicrobial compounds of the present invention.Though the utility of the compounds disclosed herein does not depend perse on their being AcpS inhibitors, the design effects toward that goalresulted, at least in part, in the discovery of the compounds of thepresent invention.

According to an embodiment, a compound, or prodrug or pharmaceuticallyacceptable salt thereof, is provided according to one of Formulas I orhas as structure as set forth in one of Formulas I:

in which E is —CH₂— or is absent whereby thiophene is directly connectedto phenyl;R₁, R₂, R₉, R₁₀, and R₁₁ (if present) are each independently selectedfrom the group consisting of hydrogen, methyl, ethyl, propyl,cyclopropyl, butyl, cyclobutyl, pentyl, hexyl, isopropryl, isobutyl,neopentyl, methoxy, and ethoxy;additionally, R₁ and R₂ may connect to form a phenyl or benzofuran ring;additionally, R₉ and R₁₀ may connect to form a phenyl or benzofuranring;R₃ and R₈ are each independently selected from the group consisting ofhydrogen, methyl, ethyl, propyl, isopropyl, chlorine, fluorine,tert-butyl, methylsulfonyl, methoxy, and ethoxy;R₄ and R₇ are each independently selected from the group consisting ofhydrogen, chlorine, methyl ester, ethyl ester, methyl, ethyl, propyl,cyclopropyl, butyl, cyclobutyl, isopropryl, isobutyl, methoxy, andethoxy;and R₅ and R₆ are each independently selected from the group consistingof hydrogen, cyclopentyl, cyclopropyl, furan, thiophene,trifluoromethyl, trifluoromethyl ether, methylthiol, formaldehyde,chlorine, fluorine, bromine, phenyl, methyl, ethyl, isopropyl, propyl,butyl, cyclobutyl, isobutyl, neopentyl, pentyl, methoxy, and ethoxy.

In some embodiments, R₁ and R₂ are either independently selected fromthe group consisting of hydrogen and methyl, or form a phenyl ringwhereby the ring system is naphthyl; R₉ and R₁₀ are either independentlyselected from the group consisting of hydrogen and methyl, or form aphenyl ring whereby the ring system is naphthyl; R₁₁ is hydrogen; R₃ andR₈ are each independently selected from the group consisting ofhydrogen, methyl, chlorine, fluorine, isopropyl, test-butyl, methoxy,and methylsulfonyl; R₄ and R₇ are each independently selected from thegroup consisting of hydrogen, methyl, chlorine, and ethyl ester; and R₅and R₆ are each independently selected from the group consisting ofmethyl, ethyl, phenyl, hydrogen, chlorine, isopropyl, cyclopentyl,bromine, cyclopropyl, trifluoromethyl, trifluoromethyl ether,methylthiol, formaldehyde, furan, and thiophene. In some embodiments, Eis absent. In some embodiments, R₁, R₂, R₄, R₇, R₉, and R₁₀ are eachhydrogen. In some embodiments, R₃ and R₈ are each chlorine; and R₅ andR₆ are selected from the group consisting of hydrogen, methyl, andisopropyl. In a preferred embodiment, the compound is DNM0488. In apreferred embodiment, the compound is DNM0548. In a preferredembodiment, the compound is DNM0606. In a preferred embodiment, thecompound is DNM0631. (These compound identifiers are given according toFIG. 1.) In a preferred embodiment, the compound is selected from thegroup consisting of those compounds listed in FIG. 1.

According to an embodiment, a compound, or prodrug or pharmaceuticallyacceptable salt thereof, is provided according to one of Formulas II:

in whichR₁, R₂, R₃, R₅, and R₁₂ are each independently selected from the groupconsisting of hydrogen, methyl, ethyl, propyl, cyclopropyl, butyl,cyclobutyl, pentyl, hexyl, isopropryl, isobutyl, neopentyl, methoxy, andethoxy;R₄, and R₁₃ are each independently selected from the group consisting ofhydrogen, methyl, ethyl, propyl, cyclopropyl, butyl, cyclobutyl, pentyl,hexyl, isopropryl, isobutyl, neopentyl, methoxy, ethoxy, anddialkylamine;additionally, R₄ and R₅ may connect to form a phenyl ring;additionally, R₁₂ and R₁₃ may connect to form a phenyl ring;R₆ and R₁₁ are each independently selected from the group consisting ofhydrogen, chlorine, fluorine, hydroxy, phenyl ether, methyl, ethyl,propyl, isopropyl, tert-butyl, methoxy, and ethoxy;R₇ and R₁₀ are each independently selected from the group consisting ofhydrogen, chlorine, methyl, ethyl, propyl, isopropyl, tert-butyl,methoxy, and ethoxy;and R₈ and R₉ are each independently selected from the group consistingof hydrogen, halogen, methyl, ethyl, propyl, isopropyl, tert-butyl,methoxy, ethoxy, naphthyl optionally substituted with methyl, and phenyloptionally substituted with chlorine, bromine, carboxylic acid, andalkyl.In some embodiments, R₆ and R₁₁ are each independently selected from thegroup consisting of hydrogen, chlorine, fluorine, hydroxy, phenyl ether,and alkyl; R₇ and R₁₀ are each independently selected from the groupconsisting of hydrogen, chlorine, and alkyl; and R₃ and R₉ are eachindependently selected from the group consisting of hydrogen, halogen,methyl, isopropyl, naphthyl optionally substituted with methyl, andphenyl optionally substituted with chlorine, bromine, carboxylic acid,and alkyl. In some embodiments, the compound is according to FormulaIIa. In some embodiments, the compound is a compound according toFormula IIb. In a preferred embodiment, the compound is selected fromthe group consisting of those compounds listed in FIG. 2.

In a preferred embodiment of the invention, a compound is selected fromthe group consisting of5-(4,5-Bis(4-chloro-2-methylphenyl)thiophen-2-yl)-1H-tetrazole(DNM0488),5-(4,5-Bis(4-methylnaphthalen-1-yl)thiophen-2-yl)-1H-tetrazole(DNM0486), 5-(4,5-Bis(4-chlorophenyl)thiophen-2-yl)-1H-tetrazole(DNM0487), Di(biphenyl-2-yl)thiophen-2-yl)-1H-tetrazole (DNM0489),5-(4,5-Bis(3-chloro-4-methylphenyl)thiophen-2-yl)-1H-tetrazole(DNM0504),5-(4,5-Bis(5-chloro-2-methylphenyl)thiophen-2-yl)-1H-tetrazole(DNM0508), 5-(4,5-Bis(3,4-dimethylphenyl)thiophen-2-yl)-1H-tetrazole(DNM0509), 5-(4,5-Bis(4-isopropylphenyl)thiophen-2-yl)-1H-tetrazole(DNM0512), 5-(4,5-Bis(2-methylphenyl)thiophen-2-yl)-1H-tetrazole(DNM0531), 5-(4,5-Bis(2-isopropylphenyl)thiophen-2-yl)-1H-tetrazole(DNM0534), 5-(4,5-Bis(2-phenoxyphenyl)thiophen-2-yl)-1H-tetrazole(DNM0536),5-(4,5-Bis(4-fluoronaphthalen-1-yl)thiophen-2-yl)-1H-tetrazole(DNM0537), 5-(4,5-Bis(2-chlorophenyl)thiophen-2-yl)-1H-tetrazole(DNM0538), 5-(4,5-Bis(2-ethylphenyl)thiophen-2-yl)-1H-tetrazole(DNM0541), 5-(4,5-Bis(dibenzo[b,d]furan-4-yl)thiophen-2-yl)-1H-tetrazole(DNM0542), 5-(4,5-Di(benzofuran-2-yl)thiophen-2-yl)-1H-tetrazole(DNM0543), 5-(4,5-Bis(2-methoxyphenyl)thiophen-2-yl)-1H-tetrazole(DNM0544), 5-(4,5-Bis(2,3-dimethoxyphenyl)thiophen-2-yl)-1H-tetrazole(DNM0545), 5-(4,5-Bis(4-tert-butylphenyl)thiophen-2-yl)-1H-tetrazole(DNM0546),5-(4,5-Bis(4-chloro-2-iso-propylphenyl)thiophen-2-yl)-1H-tetrazole(DNM0548),5-(4,5-Bis(2-(trifluoromethyl)phenyl)thiophen-2-yl)-1H-tetrazole(DNM0549),5-(4,5-Bis(2-(trifluoromethoxy)phenyl)thiophen-2-yl)-1H-tetrazole(DNM0550), 5-(4,5-Bis(2,4-dimethoxyphenyl)thiophen-2-yl)-1H-tetrazole(DNM0552), 5-(4,5-Bis(2,6-dimethoxyphenyl)thiophen-2-yl)-1H-tetrazole(DNM0553),(2,2′-(5-(1H-tetrazol-5-yl)thiophene-2,3-diyl)bis(2,1-phenylene))dimethanol(DNM0555), 2,2′-(5-(1H-tetrazol-5-yl)thiophene-2,3-diyl)dibenzaldehyde(DNM0556), 5-(4,5-Di(furan-3-yl)thiophen-2-yl)-1H-tetrazole (DNM0557),5-(4,5-Di(thiophen-3-yl)thiophen-2-yl)-1H-tetrazole (DNM0558),5-(4,5-Bis(2-(thiophen-3-yl)phenyl)thiophen-2-yl)-1H-tetrazole(DNM0559), 5-(4,5-Bis(2-(furan-3-yl)phenyl)thiophen-2-yl)-1H-tetrazole(DNM0560),5-(4,5-Bis(2-chloro-4-methylphenyl)thiophen-2-yl)-1H-tetrazole(DNM0563),5-(4,5-Bis(4-methoxy-3,5-dimethylphenyl)thiophen-2-yl)-1H-tetrazole(DNM0564),5-(4,5-Bis(3-chloro-4-methoxyphenyl)thiophen-2-yl)-1H-tetrazole(DNM0565), Diethyl3,3′-(5-(1H-tetrazol-5-yl)thiophene-2,3-diyl)dibenzoate (DNM0593),1,1′-(3,3′-(5-(1H-tetrazol-5-yl)thiophene-2,3-diyl)bis(3,1-phenylene))dibutan-1-one(DNM0599), Diethyl5,5′-(5-(1H-tetrazol-5-yl)thiophene-2,3-diyl)bis(3-hydroxybenzoate),5-(4,5-Bis(3-butylphenyl)thiophen-2-yl)-1H-tetrazole (DNM0608),5-(4,5-bis(3-(cyclopentylmethyl)phenyl)thiophen-2-yl)-1H-tetrazole(DNM0612),5-(4-(4-Chloro-2-methylphenyl)-5-(4-methylnaphthalen-1-yl)thiophen-2-yl)-1H-tetrazole(DNM0576),5-(4-(4-Chlorophenyl)-5-(4-methylnaphthalen-1-yl)thiophen-2-yl)-1H-tetrazole(DNM0572),5-(4-(5-Chlorophenyl)-4-(4-methylnaphthalen-1-yl)thiophen-2-yl)-1H-tetrazole(DNM0575),5-(4-(4-Fluorophenyl)-5-(4-(methylsulfonyl)phenyl)thiophen-2-yl)-1H-tetrazole(DNM0592), Ethyl3-(2-(4-methylnaphthalen-1-yl)-5-(1H-tetrazol-5-yl)thiophen-3-yl)benzoate(DNM0596), Ethyl3-(2-(4-chloro-2-methylphenyl)-5-(1H-tetrazol-5-yl)thiophen-3-yl)benzoate(DNM0597), 4,5-Bis(4-chloro-2-methylphenyl)-2-(1H-tetrazol-5-yl)thiazole(DNM0574), 4,5-Bis(5-chloro-2-methylphenyl)-2-(1H-tetrazol-5-yl)thiazole(DNM0567), 4,5-Bis(3-chloro-4-methylphenyl)-2-(1H-tetrazol-5-yl)thiazole(DNM0568), 4,5-Bis(4-methylnaphthalen-1-yl)-2-(1H-tetrazol-5-yl)thiazole(DNM0569), 4,5-Bis(4-chlorophenyl)-2-(1H-tetrazol-5-yl)thiazole(DNM0573), 4,5-Bis(4-fluoronaphthalen-1-yl)-2-(1H-tetrazol-5-yl)thiazole(DNM0578), 4,5-Bis(4-biphenyl)-2-(1H-tetrazol-5-yl)thiazole (DNM0581),4,5-Bis(4-t-butylphenyl)-2-(1H-tetrazol-5-yl)thiazole (DNM0582),4,5-Bis(3,4-dimethylphenyl)-2-(1H-tetrazol-5-yl)thiazole (DNM0583),4,5-Bis(4-chloro-2-isopropylphenyl)-2-(1H-tetrazol-5-yl)thiazole(DNM0584),5-(5-(4-Chlorobenzyl)-4-(4-methylnaphthalen-1-yl)thiophen-2-yl)-1H-tetrazole(DNM0577),5-(5-(4-Chlorobenzyl)-4-(4-chlorophenyl)thiophenyl)-1H-tetrazole(DNM0579),5-(5-(4-Chlorobenzyl)-4-(4-chloro-2-iso-propylphenyl)thiophenyl)-1H-tetrazole(DNM0580),5-(5-(4-Chlorobenzyl)-4-(4-fluoronaphthalen-1-yl)thiophen-2-yl)-1H-tetrazole(DNM0587),5-(4-(Biphenyl-2-yl)-5-(4-chlorobenzyl)thiophen-2-yl)-1H-tetrazole(DNM0588), Ethyl3-(2-(4-chlorobenzyl)-5-(1H-tetrazol-5-yl)thiophen-3-yl)benzoate(DNM0595),1-(3-(2-(4-Chlorobenzyl)-5-(1H-tetrazol-5-yl)thiophen-3-yl)phenyl)butan-1-one(DNM0600),5-(4-(3-Butylphenyl)-5-(4-chlorobenzyl)thiophen-2-yl)-1H-tetrazole(DNM0606), Ethyl3-(2-(4-chlorobenzyl)-5-(1H-tetrazol-5-yl)thiophen-3-yl)-5-hydroxybenzoate(DNM0609),54544-Chlorobenzyl)-4-(3-(cyclopentylmethyl)phenyl)thiophen-2-yl)-1H-tetrazole(DNM0610),3-(2-(4-Chlorobenzyl)-5-(1H-tetrazol-5-yl)thiophen-3-yl)phenol(DNM0613),5-(5-(4-Chlorobenzyl)-4-(3-methoxyphenyl)thiophen-2-yl)-1H-tetrazole(DNM0615),5-(5-(4-Chlorobenzyl)-4-(3-butoxyphenyl)thiophen-2-yl)-1H-tetrazole(DNM0616),5-(5-(4-Chlorobenzyl)-4-(3-ethoxyphenyl)thiophen-2-yl)-1H-tetrazole(DNM0617),5-(5-(4-Chlorobenzyl)-4-(3-propoxyphenyl)thiophen-2-yl)-1H-tetrazole(DNM0618), 5-(3,5-Bis(4-methylnaphthalen-1-yl)phenyl)-2H-tetrazole(DNM0461), 5-(2,5-Bis(4-methylnaphthalen-1-yl)phenyl)-1H-tetrazole(DNM0446), 5-(2,5-Bis(4-chloro-2-methylphenyl)phenyl)-1H-tetrazole(DNM0447), 5-(3,5-Bis(4-chlorophenyl)phenyl)-2H-tetrazole (DNM0470),5-(4′-Chloro-5-(4-methylnaphthalen-1-yl)biphenyl-3-yl)-1H-tetrazole(DNM0480), 5-(3,5-Bis(4-fluoronaphthalen-1-yl)phenyl)-2H-tetrazole(DNM0539), 4,5-Bis(4-chloro-2-isopropylphenyl)thiophene-2-carboxylicacid (DNM0566), 4,5-Bis(4-biphenyl)thiophene-2-carboxylic acid(DNM0497), 4,5-Bis(4-chlorophenyl)thiophene-2-carboxylic acid (DNM0498),4,5-Bis(5-chloro-2-methylphenyl)thiophene-2-carboxylic acid (DNM0501),4,5-Bis(3-chlorophenyl)thiophene-2-carboxylic acid (DNM0502),4,5-Bis(2,4-dimethylphenyl)thiophene-2-carboxylic acid (DNM0503),4,5-Bis(4-chloro2-methylphenyl)thiophene-2-carboxylic acid (DNM0561),5-(4,5-Bis(4-chloro-2-isopropylphenyl)-3-methylthiophen-2-yl)-1H-tetrazole(DNM0631),5-(4,5-bis(4-chloro-2-methylphenyl)-3-methylthiophen-2-yl)-1H-tetrazole(DNM0614),5-(4,5-Bis(4-methylnaphthalen-1-yl)-3-methylthiophen-2-yl)-1H-tetrazole(DNM0620), 5-(4,5-Bis(3-butylphenyl)-3-methylthiophen-2-yl)-1H-tetrazole(DNM0627), and5-(4,5-Bis(4-fluoronaphthalen-1-yl)-3-methylthiophen-2-yl)-1H-tetrazole(DNM0628).

According to an embodiment of the invention, a method of treatment of amicrobial infection is provided comprising administering an effectiveamount of an antimicrobial compound as described herein to a patient inneed thereof. Such a patient may be, for example, a human or othermammal that is infected with a pathogenic bacterium. In someembodiments, the microbial infection is substantially caused byGram-positive bacteria. In some embodiments, the microbial infection issubstantially caused by Enterococcus sp. In some embodiments, themicrobial infection is substantially caused by Staphyloccocus sp. Insome embodiments, the microbial infection is substantially caused byBacillus sp. In some embodiments, the microbial infection issubstantially caused by Staphylococcus epidermidis, Staphylococcusaureus (including methicillin-resistant Staphylococcus aureus [MRSA]),Enterococcus sp. (including vancomycin-resistant Enterococcus [VRE]),Streptococcus sp., or Bacillus sp. To say that a microbial infection is“substantially caused” by a particular bacterium or species thereofindicates that the given bacterial genus or species is thought to beprimarily responsible for the pathology, epidemiology, or symptomprofile of the microbial infection and does not rule out the presence ofother bacterial species (e.g., benignly colonizing bacteria). In someembodiments, the microbial infection is skin and skin structureinfection of ulcers, wound infections, diabetic foot infections,osteomyelitis, pneumonia, anthrax infection, impetigo orsecondarily-acquired traumatic lesions, gasteroenteritis, meningitis,pneumonia, gonorrhea, peptic ulcers, nosocomial infections, orblood-stream infection, among others. In some embodiments, the describedantimicrobial agents are used as medicinal compounds, for example, fortreating humans, or as veterinary compounds, for example, for treatinganimals, poultry, livestock and the like, as well as in aquaculture andagricultural applications.

According to an embodiment of the invention, a method of treatment of amicrobial infection is provided comprising administering an effectiveamount of an antimicrobial adjuvant compound as described herein and aneffective amount of an antimicrobial compound to a patient in needthereof. Such a patient may be, for example, a human or other mammalthat is infected with a pathogenic bacterium. In some embodiments, theantimicrobial adjuvant compound is DNM0487. In some embodiments, theantimicrobial adjuvant compound is DNM0488. In some embodiments, theantimicrobial adjuvant compound is DNM0548. In some embodiments, theantimicrobial adjuvant compound is itself antimicrobial to some extent.Thus combinations are possible, not only of compounds of the presentinvention with each other, but also between a compound of the presentinvention and a known antibacterial compound. In some embodiments, themicrobial infection is skin and skin structure infection of ulcers,wound infections, diabetic foot infections, osteomyelitis, pneumonia,impetigo or secondarily-acquired traumatic lesions, gasteroenteritis,meningitis, pneumonia, septicaemia, urinary tract infections, gonorrhea,peptic ulcers, nosocomial infections, blood-stream infection,brucellosis, campylobacteriosis, Cat Scratch fever, cholera,legionellosis, leptospirosis, Lyme disease, melioidosis, meningitis,pertussis, plague, salmonellosis, shigellosis, syphilis, tularemia,typhoid fever, or urinary tract infection. An effective amount of one ormore of the above-described antimicrobials may be used in thepreparation of a medicament as described above for the treatment of adisease, disorder or condition caused by a pathogenic bacteria selectedfrom the group including but by no means limited to Escherichia,Salmonella, Pseudomonas, Neisseria, Legionella, Haemophilus,Campylobacter, Helicobacter and Shigella.

In another embodiment of the invention, there is provided a method ofmanufacturing a medicament for treating a microbial infection comprisingadmixing an antimicrobial compound as described herein with a suitableexcipient.

In other embodiments, there is provided the use of an antimicrobialcompound as described herein for treating a microbial infection.

In another embodiment of the invention, there is provided a method ofmanufacturing a medicament for treating a microbial infection comprisingadmixing an antimicrobial adjuvant as described herein with a suitableantimicrobial compound.

In other embodiments, there is provided the use of an antimicrobialadjuvant as described herein for treating a microbial infection. In someembodiments, the antimicrobial adjuvant is used with or coadministeredwith an antimicrobial compound.

According to an embodiment, a pharmaceutical composition is providedcomprising a compound of the present invention and a pharmaceuticallyacceptable excipient.

The microbial infection may be substantially caused by one or moreGram-positive bacteria.

The microbial infection may comprise a Staphylococcal infection.

The microbial infection may comprise Enterococcal infection.

The microbial infection may comprise Bacillus infection.

The microbial infection may be substantially caused by a bacterialspecies selected from the group consisting of Staphylococcus aureus,Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium,Bacillus cereus, and Streptococcus sp.

Alternatively, the microbial infection is substantially caused byGram-negative bacteria.

The microbial infection may be polymicrobial.

The microbial infection may be substantially caused by Pseudomonasaeruginosa.

The antimicrobial adjuvant compound may be selected from the groupconsisting of DNM0487, DNM0488, and DNM0548.

It is of note that the antimicrobials or antimicrobial adjuvants may beprepared to be administered in a variety of ways, for example,topically, orally, intravenously, intramuscularly, subcutaneously,intraperitoneally, intranasally or by local or systemic intravascularinfusion using means known in the art and as discussed below. Compoundsof the present invention may be used, for example, to treat infectionscaused by Gram negative bacteria such as E. coli and/or Gram positivebacteria such as S. aureus. In some embodiments, compounds haveantimicrobial effects against antibiotic-resistant strains such as, forexample, methicillin-resistant S. aureus (MRSA). In some embodiments,compounds have antimicrobial effects against Mycobacterium tuberculosis.

The antimicrobial or antimicrobial adjuvant compounds may be arranged tobe delivered at a concentration of about 1 nM to about 50 mM; or 10 nMto about 50 mM; or 100 nM to about 50 mM; or 1 μM to about 50 mM; or 10μM to 50 mM or 100 μM to 50 mM. As will be appreciated by one of skillin the art, this may be the “effective amount,” that is, a sufficientdosage is administered such that a concentration within one of theenvisioned ranges is attained at the required site. In someantimicrobial embodiments, the effective amount will be informed atleast in part by the minimum inhibitory concentration of the compoundrequired to exhibit bacteriostatic or bacteriocidal effects against thepathogen of interest. In some antimicrobial adjuvant embodiments, theeffective amount will be informed at least in part by the approximateminimum concentration required to produce the desired adjuvant effectwith a predetermined known antibiotic against the pathogen of interest.In some embodiments, the effective amount will be calibrated so as toproduce a serum level of over ten times the MIC, or over five times theMIC, or over three times the MIC, or at the MIC in a subject sufferingfrom an infection. In some embodiments, the effective amount will becalibrated so as to produce an in situ concentration of over ten timesthe MIC, or over five time the MIC, or over three times the MIC, or atthe MIC.

An effective amount of one of the compounds of the invention can beemployed in pure form or, where such forms exist, in pharmaceuticallyacceptable salt form. Alternatively, the compound can be administered asa pharmaceutical composition containing the compound of interest incombination with one or more pharmaceutically acceptable carriers. Itwill be understood, however, that the total daily usage of the compoundsand compositions of the invention will be decided by the attendingphysician within the scope of sound medical judgment. The specificeffective dose level for any particular patient will depend upon avariety of factors including the disorder being treated and the severityof the disorder; activity of the specific compound employed; thespecific composition employed; the age, body weight, general health, sexand diet of the patient; the time of administration, route ofadministration, and rate of excretion of the specific compound employed;the duration of the treatment; the risk/benefit ratio; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts. For example, it is wellwithin the skill of the art to start doses of the compound at levelslower than required to achieve the desired therapeutic effect and togradually increase the dosage until the desired effect is achieved.

The total daily dose of the compounds of the present invention asadministered to a human or lower animal may range from about 0.0003 toabout 30 mg/kg of body weight. For purposes of oral administration, morepreferable doses can be in the range of from about 0.0003 to about 1mg/kg body weight. If desired, the effective daily dose can be dividedinto multiple doses for purposes of administration; consequently, singledose compositions may contain such amounts or submultiples thereof tomake up the daily dose. For oral administration, the compositions of theinvention are preferably provided in the form of tablets containingabout 1.0, about 5.0, about 10.0, about 15.0, about 25.0, about 50.0,about 100, about 250, or about 500 milligrams of the active ingredient.

For best results, whether a particular compound is antimicrobial or anantimicrobial adjuvant with respect to the pathogen of interest (and inthe case of adjuvants, with respect to the antimicrobial compound beingused) would generally be ascertained by a number of microbiologicalmethods known in the art, including for example, the methods disclosedin the Examples. It is well understood in the art that while every“antimicrobial” compound has an antimicrobial effect against at leastone pathogen at one or more effective amounts, antimicrobial compoundsvary as to their spectrum and/or potency of activity. Moreover, whileevery “antimicrobial adjuvant” compound has an adjuvant effect inconjunction with at least one antimicrobial compound against at leastone pathogen at one or more effective amounts of both antimicrobialcompound and adjuvant, antimicrobial adjuvant compounds vary as to theirspectrum, potency of activity, and/or compatibility with particularantimicrobial compounds. As will be apparent to one knowledgeable in theart, the total dosage will vary according to many factors, including butby no means limited to the weight, age and condition of the individualor patient.

In some embodiments, one or more of the antimicrobial compounds may beco-administered with one or more known antibiotics. In some embodiments,one or more of the antimicrobial adjuvant compounds my beco-administered with one or more antimicrobial compounds; in which casethe total effective amount of antimicrobial compound may be less thanwould otherwise be required in the absence of antimicrobial adjuvant,e.g. ˜8 times less, or ˜16 times less, or ˜32 times less, or ˜64 timesless, or ˜125 times less, or ˜250 times less. In some embodiments, theantimicrobial adjuvant compounds are not themselves antimicrobial. Insome embodiments, the antimicrobial adjuvant compounds are themselvesantimicrobial. In some embodiments, one or more of the antimicrobialadjuvant compounds may be combined with one or more antimicrobialcompounds in a single dosage form. In some embodiments, theantimicrobial compound is an antimicrobial compound of the presentinvention. In some embodiments, the antimicrobial compound is a knownantimicrobial compound such as, for example, almecillin, amdinocillin,amikacin, amoxicillin, amphomycin, amphotericin B, ampicillin,azacitidine, azaserine, azithromycin, azlocillin, aztreonam,artemisinin, allopurinol, amicacin, aminoglycosides, amphotericin B,ampicillin, ansamycins, anthracyclines, antimycotics, azithromycin,bacampicillin, bacitracin, benzyl polylysine, bleomycin, brefeldin A,butoconazole, candicidin, capreomycin, carbenicillin, cefaclor,cefadroxil, cefamandole, cefazoline, cefdinir, cefepime, cefixime,cefinenoxime, cefinetazole, cefodizime, cefonicid, cefoperazone,ceforanide, cefotaxime, cefotetan, cefotiam, cefoxitin, cefpiramide,cefpodoxime, cefprozil, cefsulodin, ceftazidime, ceftibuten,ceftizoxime, ceftriaxone, cefuroxime, cephacetrile, cephalexin,cephaloglycin, cephaloridine, cephalothin, cephapirin, cephradine,chloramphenicol, cilastatin, cinnamycin, ciprofloxacin, clarithromycin,clavulanic acid, clindamycin, clioquinol, cloxacillin, colistimethate,colistin, cyclacillin, cycloserine, cyclosporine, cyclo-(Leu-Pro),camptothecin, cefataxime, cephalexin, cephalosporins, chalcomycin,chartreusin, chlorotetracyclines, chlorothricin, chrymutasins,chrysomicin M, chrysomicin V, clomocyclines, dactinomycin, dalbavancin,dalfopristin, daptomycin, daunorubicin, demeclocycline, detorubicin,dicloxacillin, dihydrostreptomycin, dirithromycin, doxorubicin,doxycycline, ellipticines, elsamicin, epirubicin, erythromycin,eveminomycin, filipins, fluconazoles, fungichromins, fusidic acid,floxacillin, fosfomycin, gentamycin, gilvocarin, griseofulvin,griseoviridin, guamecyclines, gemifloxacin, gramicidin, hetacillin,idarubicin, imipenem, iseganan, ivermectin, ilosamides, itraconazoles,kanamycin, laspartomycin, linezolid, loracarbef, lankamycin, lincomycin,magainin, meclocycline, meropenem, methacycline, mezlocillin,minocycline, mitomycin, moenomycin, moxalactam, moxifloxacin,mycophenolic acid, macrolides, methicillins, mitoxantrone, nafcillin,natamycin, neomycin, netilmicin, niphimycin, nitrofurantoin, novobiocin,nalidixic acid, norfloxin, nystatin, nystatins, ofloxacin, oleanomycin,oxytetracyline, paromomycin, penicillamine, phenethicillin,piperacillin, plicamycin, pristinamycin, pecilocin, penicillins,pesticides, phosphomycin, pimarcin, platensimycin, polyenes, polymyxinB, polymyxin E, quinupristin, quinolones, ravidomycin, reserpines,rifamycin, ristocetins A and B, rifabutin, rifampin, rifamycin,rolitetracycline, sisomycin, spiramycin, spironolactone, sulfacetamidesodium, sulphonamide, spectrinomycin, streptomycin, streptozocin,sulbactam, sultamicillin, tacrolimus, tazobactam, teicoplanin,telithromycin, teramycins, tetracyclines, thiamphenicols, thiolutins,tobramycin, tyrothricin, ticarcillin, tigecycline, tobramycin,troleandomycin, tunicamycin, tyrthricin, vancomycin, vidarabine,viomycin, virginiamycin, and wortmannins; the presence of a plural itemin the foregoing list meaning to refer to one or more members of afamily of antibiotics known in the art by that name. Which compound orcompounds should be co-administered or compounded in combination withcompounds of the present invention depends on a number of factors,including but not necessarily limited to the efficacy of the agent oragents in the absence of antimicrobial adjuvant compounds, the mechanismof action of the compound(s), the identity of the pathogen causing orpotentiating the sick condition, and/or the severity of the sickcondition in the subject.

The pharmaceutical compositions can be formulated for oraladministration in solid or liquid form, for parenteral intravenous,subcutaneous, intramuscular, intraperitoneal, intra-arterial, orintradermal injection, for or for vaginal, nasal, topical, or rectaladministration. Pharmaceutical compositions of the present inventionsuitable for oral administration can be presented as discrete dosageforms, e.g., tablets, chewable tablets, caplets, capsules, liquids, andflavored syrups. Such dosage forms contain predetermined amounts ofactive ingredients, and may be prepared by methods of pharmacy wellknown to those skilled in the art. See generally, Remington'sPharmaceutical Sciences, 18th ed., Mack Publishing, Easton Pa. (1990).

Parenteral dosage forms can be administered to patients by variousroutes including subcutaneous, intravenous (including bolus injection),intramuscular, and intraarterial. Because their administration typicallybypasses patients' natural defenses against contaminants, parenteraldosage forms are specifically sterile or capable of being sterilizedprior to administration to a patient. Examples of parenteral dosageforms include solutions ready for injection, dry products ready to bedissolved or suspended in a pharmaceutically acceptable vehicle forinjection, suspensions ready for injection, and emulsions.Pharmaceutical compositions for parenteral injection comprisepharmaceutically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents, solventsor vehicles include water, ethanol, polyols (propylene glycol,polyethylene glycol, glycerol, and the like, and suitable mixturesthereof), vegetable oils (such as olive oil) and injectable organicesters such as ethyl oleate, or suitable mixtures thereof. Suitablefluidity of the composition may be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersions, and by the use of surfactants.These compositions may also contain adjuvants such as preservativeagents, wetting agents, emulsifying agents, and dispersing agents.Prevention of the action of microorganisms may be ensured by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, and the like. It may also bedesirable to include isotonic agents, for example, sugars, sodiumchloride and the like. Prolonged absorption of the injectablepharmaceutical form may be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is oftendesirable to slow the absorption of the drug from subcutaneous orintramuscular injection. This may be accomplished by the use of a liquidsuspension of crystalline or amorphous material with poor watersolubility. The rate of absorption of the drug then depends upon itsrate of dissolution which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of a parenterallyadministered drug form is accomplished by dissolving or suspending thedrug in an oil vehicle.

Suspensions, in addition to the active compounds, may contain suspendingagents, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar, tragacanth, and mixtures thereof.If desired, and for more effective distribution, the compounds of theinvention can be incorporated into slow-release or targeted-deliverysystems such as polymer matrices, liposomes, and microspheres. They maybe sterilized, for example, by filtration through a bacteria-retainingfilter or by incorporation of sterilizing agents in the form of sterilesolid compositions, which may be dissolved in sterile water or someother sterile injectable medium immediately before use.

Injectable depot forms are made by forming microencapsulated matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending upon the ratio of drug to polymer and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations also are prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissues. The injectable formulations can be sterilized, forexample, by filtration through a bacterial-retaining filter or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable medium just prior to use.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic, parenterally acceptablediluent or solvent such as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, one or morecompounds of the invention is mixed with at least one inertpharmaceutically acceptable carrier such as sodium citrate or dicalciumphosphate and/or a) fillers or extenders such as starches, lactose,sucrose, glucose, mannitol, and salicylic acid; b) binders such ascarboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia; c) humectants such as glycerol; d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; e) solutionretarding agents such as paraffin; f) absorption accelerators such asquaternary ammonium compounds; g) wetting agents such as cetyl alcoholand glycerol monostearate; h) absorbents such as kaolin and bentoniteclay; and i) lubricants such as talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof. In the case of capsules, tablets and pills, the dosageform may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using lactose or milk sugar aswell as high molecular weight polyethylene glycols. The solid dosageforms of tablets, dragees, capsules, pills, and granules can be preparedwith coatings and shells such as enteric coatings and other coatingswell known in the pharmaceutical formulating art. They may optionallycontain opacifying agents and can also be of a composition that theyrelease the active ingredient(s) only, or preferentially, in a certainpart of the intestinal tract in a delayed manner. Examples of materialswhich can be useful for delaying release of the active agent can includepolymeric substances and waxes.

Dosage forms for topical administration may include powders, sprays,ointments and inhalants. A compound of the present invention can bemixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives, buffers or propellants which maybe required. Opthalmic formulations, eye ointments, powders andsolutions are contemplated as being within the scope of this invention.Aqueous liquid compositions comprising compounds of the invention alsoare contemplated.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active compounds, the liquid dosage formsmay contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents. Dosage forms for topical or transdermaladministration of a compound of this invention include ointments,pastes, creams, lotions, gels, powders, solutions, sprays, inhalants orpatches. A desired compound of the invention is admixed under sterileconditions with a pharmaceutically acceptable carrier and any neededpreservatives or buffers as may be required. Ophthalmic formulation, eardrops, eye ointments, powders and solutions are also contemplated asbeing within the scope of this invention. The ointments, pastes, creamsand gels may contain, in addition to an active compound of thisinvention, animal and vegetable fats, oils, waxes, paraffins, starch,tragacanth, cellulose derivatives, polyethylene glycols, silicones,bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the compounds of thisinvention, lactose, talc, silicic acid, aluminum hydroxide, calciumsilicates and polyamide powder, or mixtures of these substances. Sprayscan additionally contain customary propellants such aschlorofluorohydrocarbons.

Compounds of the invention may also be administered in the form ofliposomes. As is known in the art, liposomes are generally derived fromphospholipids or other lipid substances. Liposomes are formed by mono-or multi-lamellar hydrated liquid crystals that are dispersed in anaqueous medium. Any non-toxic, physiologically acceptable andmetabolizable lipid capable of forming liposomes may be used. Thepresent compositions in liposome form may contain, in addition to thecompounds of the invention, stabilizers, preservatives, and the like.The preferred lipids are the natural and synthetic phospholipids andphosphatidylcholines (lecithins) used separately or together. Methods toform liposomes are known in the art. See, for example, Prescott, Ed.,Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y.,(1976), p 33 et seq.

In some embodiments, one or more of the antimicrobials or antimicrobialadjuvants at concentrations or dosages discussed above may be combinedwith a pharmaceutically or pharmacologically acceptable carrier,excipient or diluent, either biodegradable or non-biodegradable.Exemplary examples of carriers include, but are by no means limited to,for example, poly(ethylene-vinyl acetate), copolymers of lactic acid andglycolic acid, poly(lactic acid), gelatin, collagen matrices,polysaccharides, poly(D,L lactide), poly(malic acid),poly(caprolactone), celluloses, albumin, starch, casein, dextran,polyesters, ethanol, mathacrylate, polyurethane, polyethylene, vinylpolymers, glycols, mixtures thereof and the like. Standard excipientsinclude gelatin, casein, lecithin, gum acacia, cholesterol, tragacanth,stearic acid, benzalkonium chloride, calcium stearate, glycerylmonostearate, cetostearyl alcohol, cetomacrogol emulsifying wax,sorbitan esters, polyoxyethylene alkyl ethers, polyoxyethylene castoroil derivatives, polyoxyethylene sorbitan fatty acid esters,polyethylene glycols, polyoxyethylene stearates, colloidol silicondioxide, phosphates, sodium dodecylsulfate, carboxymethylcellulosecalcium, carboxymethylcellulose sodium, methylcellulose,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethycellulose phthalate, noncrystalline cellulose,magnesium aluminum silicate, triethanolamine, polyvinyl alcohol,polyvinylpyrrolidone, sugars and starches.

As will be apparent to one knowledgeable in the art, specific carriersand carrier combinations known in the art may be selected based on theirproperties and release characteristics in view of the intended use.Specifically, the carrier may be pH-sensitive, thermo-sensitive,thermo-gelling, arranged for sustained release or a quick burst. In someembodiments, carriers of different classes may be used in combinationfor multiple effects, for example, a quick burst followed by sustainedrelease.

In other embodiments, one or more of the antimicrobials or antimicrobialadjuvants at concentrations or dosages described above may beencapsulated for delivery. Specifically, the compounds may beencapsulated in biodegradable microspheres, microcapsules,microparticles, or nanospheres. The delivery vehicles may be composedof, for example, hyaluronic acid, polyethylene glycol, poly(lacticacid), gelatin, poly(E-caprolactone), or a poly(lactic-glycolic) acidpolymer. Combinations may also be used, as, for example, gelatinnanospheres may be coated with a polymer of poly(lactic-glycolic) acid.As will be apparent to one knowledgeable in the art, these and othersuitable delivery vehicles may be prepared according to protocols knownin the art and utilized for delivery of the compounds.

It is of note that the above described antimicrobials may be combinedwith permeation enhancers known in the art for improving delivery.Examples of permeation enhancers include, but are by no means limited tothose compounds described in U.S. Pat. Nos. 3,472,931; 3,527,864;3,896,238; 3,903,256; 3,952,099; 4,046,886; 4,130,643; 4,130,667;4,299,826; 4,335,115; 4,343,798; 4,379,454; 4,405,616; 4,746,515;4,788,062; 4,820,720; 4,863,738; 4,863,970; and 5,378,730; British Pat.No. 1,011,949; and Idson, 1975, J. Pharm. Sci. 64:901-924.

A “pharmaceutically acceptable salt” includes a salt that retains thedesired biological activity of the parent antimicrobial or antimicrobialadjuvant compound and does not impart any undesired toxicologicaleffects. Examples of such salts are salts of acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, andthe like; acetic acid, oxalic acid, tartaric acid, succinic acid, malicacid, benzoic acid, pamoic acid, alginic acid, methanesulfonic acid,naphthalenesulfonic acid, and the like. Also included are salts ofcations such as sodium, potassium, lithium, zinc, copper, barium,bismuth, calcium, and the like; or organic cations such astrialkylammonium. Combinations of the above salts are also useful. It isto be understood that a compound disclosed herein in a salt-free formand analogous compound in a pharmaceutically acceptable salt form areboth compounds of the present invention. Additionally, prodrugs, e.g.esters of the compounds disclosed herein, are compounds of the presentinvention. The preparation and use of acid addition salts, carboxylatesalts, amino acid addition salts, and zwitterion salts of compounds ofthe present invention may also be considered pharmaceutically acceptableif they are, within the scope of sound medical judgment, suitable foruse in contact with the tissues of humans and lower animals withoutundue toxicity, irritation, allergic response, and the like, arecommensurate with a reasonable benefit/risk ratio, and are effective fortheir intended use. Such salts may also include various solvates andhydrates of the compound of the present invention.

In some embodiments, the described antimicrobial compounds used asmedicinal compounds, for example, for treating humans, or as veterinarycompounds, for example, for treating animals, poultry, livestock and thelike, as well as in aquaculture and agricultural applications.

While various embodiments of the invention have been described above, itwill be recognized and understood that modifications may be madetherein, and the appended claims are intended to cover all suchmodifications which may fall within the spirit and scope of theinvention.

Certain embodiments of the invention will now be illustrated usingexamples.

Example 1 Synthesis of DNM0488 and Analogues

4,5-Dibromothiophene-2-carboxamide

Thiophene-2-carbonitrile (3.27 g, 30.0 mmol) was dissolved in a mixtureof H₂SO₄ (10 mL) and TFA (20 mL). NBS (11.75 g, 66.0 mmol) was added inportion in 20 min. After complete addition, the reaction mixture wasfurther stirred for 4 h, and then poured onto 200 g of crushed ice. Awhite solid was formed, which was collected, washed with water, anddried together with phosphorous pentoxide under vacuum to afford 8.50 g(99%) of product.

4,5-Dibromothiophene-2-carbonitrile

An oven-dried round-bottomed flask was charged with4,5-dibromothiophene-2-carboxamide (4.28 g, 15.0 mmol) and 20 mL of DMFunder an argon atmosphere. The solution was cooled on an ice-water bath,and cyanuric chloride (1.81 g, 9.8 mmol) was then added in one portion.After stirring at 0° C. for 1 h, the reaction mixture was warmed to roomtemperature, and stirred for a further 3 h. 100 mL of water was added. Awhite solid was formed, which was collected through suction filtration,washed with water, and dried together with phosphorous pentoxide undervacuum to afford 3.70 g (92%) of product.

4,5-Bis(4-chloro-2-methylphenyl)thiophene-2-carbonitrile

A round-bottomed flask was charged with4,5-dibromothiophene-2-carbonitrile (534 mg, 2.00 mmol),4-chloro-2-methylphenylboronic acid (818 mg, 4.80 mmol) and Pd(PPh₃)₄(136 mg, 0.10 mmol). After degassed, dioxane (10 mL) and aqueous sodiumcarbonate (5 mL, 2M, 10 mmol) was added. The reaction mixture was heatedto 90° C. The progress of the reaction was monitored by TLC. After thereaction was complete, 50 mL of water was added, and the reactionmixture was extracted with ethyl acetate. The organic phase was driedover anhydrous sodium sulphate and then concentrated. The residue waspurified by flash chromatography (hexane:CH₂Cl₂=2:1). 0.60 g (84%) ofproduct was obtained as clear oil.

5-(4,5-Bis(4-chloro-2-methylphenyl)thiophen-2-yl)₄H-tetrazole (DNM0488)

A round-bottomed flask was charged with4,5-bis(4-chloro-2-methylphenyl)thiophene-2-carbonitrile (600 mg, 1.67mmol), zinc bromide (945 mg, 4.20 mmol) and sodium azide (273 mg, 4.20mmol). After degassed, DMF (5 mL) was added. The reaction mixture washeated to 110° C. and stirred at this temperature until complete. Thereaction was cooled to rt, and 30 mL of 0.1 N aqueous HCl was added. Thereaction mixture was extracted with ethyl acetate. The organic phase wasdried over anhydrous sodium sulphate and then concentrated. The residuewas purified by flash chromatography (hexane:EtOAc:AcOH=30:10:1). 603 mg(90%) of product was obtained as a white solid, ¹H NMR (DMSO, 500 MHz) δ7.82 (s, 1H), 7.36 (d, J=2.04 Hz, 1H), 7.34 (d, J=1.94 Hz, 1H), 7.31 (d,J=8.25 Hz, 1H), 7.28 (dd, J₁=8.26 Hz, J₂=1.99 Hz, 1H), 7.17 (dd, J₁=8.20Hz, J₂=2.05 Hz, 1H), 7.02 (d, J=8.28 Hz, 1H), 2.17 (s, 3H), 2.03 (s,3H); ¹³C NMR (DMSO, 125 MHz) δ 140.58, 139.14, 139.12, 138.19, 133.59,133.39, 132.94, 132.47, 131.71, 131.17, 130.57, 130.17, 130.11, 126.00,125.80, 19.69, 19.57.

The following compounds were also prepared using an analogous method:

5-(4,5-Bis(4-methylnaphthalen-1-yl)thiophen-2-yl)-1H-tetrazole (DNM0486)

¹H NMR (DMSO, 500 MHz) δ 8.00 (d, J=8.49 Hz, 1H), 7.98-7.90 (m, 3H),7.88 (d, J=8.49 Hz, 1H), 7.57-7.50 (m, 2H), 7.50-7.42 (m, 2H), 7.33 (t,J=7.47 Hz, 1H), 7.29 (d, J=7.36 Hz, 1H), 7.19 (d, J=7.22 Hz, 1H), 7.13(d, J=7.22 Hz, 1H), 2.57 (s, 3H), 2.52 (s, 3H); ¹³C NMR (DMSO, 125 MHz)δ 141.36, 135.65, 134.20, 132.18, 132.13, 131.98, 131.68, 131.30,130.95, 129.21, 127.85, 127.50, 126.30, 126.22, 126.12, 125.95, 125.89,125.81, 125.75, 124.55, 124.50, 19.07, 18.97.

5-(4,5-Bis(4-chlorophenyl)thiophen-2-yl)-1H-tetrazole (DNM0487)

¹H NMR (DMSO, 500 MHz) δ 7.90 (s, 1H), 7.50-7.45 (m, 4H), 7.38-7.31 (m,4H); ¹³C NMR (DMSO, 125 MHz) δ 140.14, 137.85, 133.55, 133.45, 132.63,131.58, 131.15, 130.74, 130.54, 129.19, 128.94, 124.74.

5-(4,5-Di(biphenyl-2-yl)thiophen-2-yl)-1H-tetrazole (DNM0489)

¹H NMR (DMSO, 500 MHz) δ 7.48 (s, 1H), 7.35 (td, J₁=7.55 Hz, J₂=1.00 Hz,1H), 7.30 (td, J₁=7.55 Hz, J₂=1.00 Hz, 1H), 7.21-7.03 (m, 10H),6.72-6.65 (m, 2H), 6.65-6.58 (m, 3H), 6.55 (d, J=7.45 Hz, 1H); ¹³C NMR(DMSO, 125 MHz) δ 141.91, 140.83, 140.46, 140.19, 139.33, 132.94,132.02, 131.21, 130.32, 130.23, 130.20, 129.88, 128.63, 128.46, 128.45,127.95, 127.88, 127.70, 127.45, 127.34, 126.61, 126.34.

5-(4,5-Bis(3-chloro-4-methylphenyl)thiophen-2-yl)-1H-tetrazole (DNM0504)

¹H NMR (DMSO, 500 MHz) δ 7.89 (s, 1H), 7.41 (d, J=1.70 Hz, 1H), 7.39 (d,J=1.75 Hz, 1H), 7.37 (d, J=8.05 Hz, 1H), 7.35 (d, J=8.00 Hz, 1H), 7.18(dd, J₁=7.85 Hz, J₂=1.80 Hz, 1H), 7.12 (dd, J=7.83 Hz, J₂=1.73 Hz, 1H),2.34 (s, 6H); ¹³C NMR (DMSO, 125 MHz) δ 139.77, 137.40, 136.19, 135.06,134.02, 133.71, 133.55, 131.80, 131.68, 131.60, 131.54, 128.79, 128.69,127.71, 127.43, 19.38, 19.32.

5-(4,5-Bis(5-chloro-2-methylphenyl)thiophen-2-yl)-1H-tetrazole (DNM0508)

¹H NMR (DMSO, 500 MHz) δ 7.84 (s, 1H), 7.41 (d, J=2.25 Hz, 1H), 7.36(dd, J₁=8.23 Hz, J₂=2.28 Hz, 1H), 7.31-7.23 (m, 3H), 7.11 (s, 1H), 2.13(s, 3H), 2.01 (s, 3H); ¹³C NMR (DMSO, 125 MHz) δ 140.36, 139.01, 136.27,135.74, 134.71, 133.43, 132.26, 132.23, 131.07, 130.59, 130.15, 130.01,129.56, 128.95, 19.26, 19.16.

5-(4,5-Bis(3,4-dimethylphenyl)thiophen-2-yl)-1H-tetrazole (DNM0509)

¹H NMR (DMSO, 500 MHz) δ 7.84 (s, 1H), 7.21 (s, 1H), 7.19 (s, 1H),7.12-7.06 (m, 2H), 6.98 (dd, J₁=8.28 Hz, J₂=1.63 Hz, 1H), 6.94 (dd,J₁=7.67 Hz, J₂=1.48 Hz, 1H), 2.22 (s, 6H), 2.20 (s, 3H), 2.19 (s, 3H);¹³C NMR (DMSO, 125 MHz) δ 141.17, 138.26, 136.90, 136.80, 136.57,135.65, 132.51, 131.95, 130.14, 129.86, 129.68, 129.65, 129.54, 126.21,126.06, 19.41, 19.33, 19.14, 19.11.

5-(4,5-Bis(4-isopropylphenyl)thiophen-2-yl)-1H-tetrazole (DNM0512)

¹H NMR (DMSO, 500 MHz) δ 7.84 (s, 1H), 7.30-7.23 (m, 8H), 2.90 (sep,J=6.88 Hz, 1H), 1.24-1.18 (m, 12H); ¹³C NMR (DMSO, 125 MHz) δ 148.87,147.77, 141.11, 138.25, 132.48, 132.01, 130.15, 128.75, 128.55, 126.93,126.67, 33.11, 33.08, 23.76, 23.65.

5-(4,5-Bis(2-methylphenyl)thiophen-2-yl)-1H-tetrazole (DNM0531)

¹H NMR (DMSO, 500 MHz) δ 7.81 (s, 1H), 7.33-7.16 (m, 6H), 7.02 (t,J=7.35 Hz, 1H), 7.02 (t, J=7.44 Hz, 1H), 2.16 (s, 3H), 2.02 (s, 3H); ¹³CNMR (DMSO, 125 MHz) δ 141.77, 140.03, 136.57, 135.51, 134.73, 131.87,131.48, 131.26, 130.42, 130.39, 130.10, 128.93, 127.89, 125.89, 125.72,19.91, 19.75.

5-(4,5-Bis(2-isopropylphenyl)thiophen-2-yl)-1H-tetrazole (DNM0534)

¹H NMR (CDCl₃, 500 MHz) δ 7.75 (s, 1H), 7.29-7.19 (m, 5H), 7.14-7.08 (m,1H), 7.07-7.00 (m, 2H), 3.10-2.95 (m, 2H), 1.04 (s, 6H), 0.95 (d, J=6.80Hz, 6H); ¹³C NMR (CDCl₃, 125 MHz) δ 148.48, 147.51, 140.43, 132.28,131.09, 129.46, 128.43, 126.23, 126.15, 125.72, 125.66, 30.36, 10.15,24.46.

5-(4,5-Bis(2-phenoxyphenyl)thiophen-2-yl)-1H-tetrazole (DNM0536)

¹H NMR (DMSO, 500 MHz) δ 7.91 (s, 1H), 7.39-7.22 (m, 8H), 7.16-7.05 (m,4H), 6.79 (d, J=7.90 Hz, 1H), 6.75 (d, J=7.65 Hz, 1H), 6.66-6.61 (m,4H); ¹³C NMR (DMSO, 125 MHz) δ 155.83, 155.76, 154.04, 153.87, 138.02,136.48, 131.75, 131.63, 131.33, 130.42, 129.91, 129.86, 129.50, 126.43,124.08, 123.68, 123.64, 123.46, 123.24, 118.95, 118.70, 118.37, 117.81.

5-(4,5-Bis(4-fluoronaphthalen-1-yl)thiophen-2-yl)-1H-tetrazole (DNM0537)

¹H NMR (DMSO, 500 MHz) δ 8.05-7.95 (m, 4H), 7.85 (d, J=8.50 Hz, 1H),7.66-7.56 (m, 3H), 7.53 (t, J=7.53 Hz, 1H), 7.40 (t, J=7.55 Hz, 1H),7.34-7.26 (m, 2H), 7.15 (dd, J₁=10.55 Hz, J₂=8.00 Hz, 1H); ¹³C NMR(DMSO, 125 MHz) δ 159.20, 158.48, 157.19, 156.48, 140.42, 139.24,132.83, 132.79, 132.44, 132.40, 132.00, 129.91, 129.84, 128.92, 128.89,127.97, 127.90, 127.80, 127.75, 127.02, 126.85, 125.89, 125.85, 125.53,125.51, 125.41, 125.39, 122.86, 122.75, 122.73, 122.62, 120.25, 120.23,120.21, 120.19, 109.42, 109.28, 109.26, 109.12.

5-(4,5-Bis(2-chlorophenyl)thiophen-2-yl)-1H-tetrazole (DNM0538)

¹H NMR (CDCl₃, 500 MHz) δ 8.07 (s, 1H), 7.36 (d, J=8.05 Hz, 1H), 7.31(d, J=7.91 Hz, 1H), 7.29-7.22 (m, 2H), 7.18 (dt, J₁=1.06 Hz, J₂=7.50 Hz,1H), 7.15-7.09 (m, 1H), 7.08-7.01 (m, 2H); ¹³C NMR (CDCl₃, 125 MHz) δ142.02, 139.64, 134.55, 134.27, 133.64, 133.21, 132.59, 132.11, 131.78,130.56, 130.40, 130.23, 129.57, 127.08, 126.97, 124.30.

5-(4,5-Bis(2-ethylphenyl)thiophen-2-yl)-1H-tetrazole (DNM0541)

¹H NMR (DMSO, 500 MHz) δ 7.77 (s, 1H), 7.33-7.20 (m, 5H), 7.17 (t,J=7.43 Hz, 1H), 7.08 (dt, J₁=1.10 Hz, J₂=7.33 Hz, 1H), 7.03 (d, J=7.50Hz, 1H), 2.50 (q, J=7.50 Hz, 2H), 2.43 (q, J=7.50 Hz, 2H), 1.06 (t,J=7.50 Hz, 3H), 0.96 (t, J=7.50 Hz, 3H); ¹³C NMR (DMSO, 125 MHz) δ142.74, 141.68, 141.58, 139.90, 134.00, 131.53, 131.48, 130.97, 130.33,129.16, 128.69, 128.45, 128.11, 125.71, 125.56, 123.74, 25.51, 25.38,15.09, 15.01.

5-(4,5-Bis(dibenzo[b,d]furan-4-yl)thiophen-2-yl)-1H-tetrazole (DNM0542)

¹H NMR (DMSO, 500 MHz) δ 8.26 (s, 1H), 8.15-8.07 (m, 3H), 8.04 (dd,J₁=7.63 Hz, J₂=0.98 Hz, 1H), 7.50-7.40 (m, 4H), 7.40-7.34 (m, 2H),7.34-7.22 (m, 4H); ¹³C NMR (DMSO, 125 MHz) δ 155.30, 155.22, 152.65,152.36, 137.03, 135.52, 131.54, 128.15, 127.93, 127.75, 127.68, 124.22,124.05, 123.36, 123.34, 123.20, 123.16, 123.12, 121.82, 121.36, 121.28,120.90, 119.86, 117.22, 111.38, 111.25.

5-(4,5-Di(benzofuran-2-yl)thiophen-2-yl)-1H-tetrazole (DNM0543)

¹H NMR (DMSO, 500 MHz) δ 8.23 (s, 1H), 7.74 (d, J=7.75 Hz, 2H), 7.67 (t,J=7.28 Hz, 2H), 7.49 (s, 1H), 7.45-7.37 (m, 3H), 7.33 (dt, J₁=2.20 Hz,J₂=7.40 Hz, 2H); ¹³C NMR (DMSO, 125 MHz) δ 154.12, 153.91, 149.67,147.67, 130.34, 130.05, 128.48, 128.36, 128.21, 125.92, 125.38, 123.75,123.53, 121.93, 121.68, 111.27, 111.26, 107.06, 106.01.

5-(4,5-Bis(2-methoxyphenyl)thiophen-2-yl)-1H-tetrazole (DNM0544)

¹H NMR (DMSO, 500 MHz) δ 7.82 (s, 1H), 7.35-7.28 (m, 2H), 7.10 (dd,J₁=7.55 Hz, J₂=1.50 Hz, 1H), 7.04 (d, J=8.35 Hz, 2H), 6.98 (dd, J₁=7.50Hz, J₂=1.35 Hz, 1H), 6.90-6.80 (m, 2H), 3.62 (s, 3H), 3.59 (s, 3H); ¹³CNMR (DMSO, 125 MHz) δ 156.29, 156.25, 138.75, 137.01, 131.72, 130.91,130.30, 130.09, 129.16, 124.59, 121.73, 120.37, 120.29, 111.85, 111.60,55.28, 55.22.

5-(4,5-Bis(2,3-dimethoxyphenyl)thiophen-2-yl)-1H-tetrazole (DNM0545)

¹H NMR (DMSO, 500 MHz) δ 7.82 (s, 1H), 7.03 (dt, J₁=1.20 Hz, J₂=8.00 Hz,2H), 6.98 (d, J=7.70 Hz, 1H), 6.94 (d, J=8.00 Hz, 1H), 6.67 (dd, J₁=7.75Hz, J₂=1.40 Hz, 1H), 6.63 (dd, J₁=7.58 Hz, J₂=1.58 Hz, 1H), 3.82 (s,6H), 3.63 (s, 3H), 3.57 (s, 3H); ¹³C NMR (DMSO, 125 MHz) δ 152.73,146.13, 146.07, 138.01, 136.68, 131.51, 129.53, 126.64, 123.88, 122.59,122.43, 113.24, 112.63, 60.12, 59.96, 55.72, 55.67.

5-(4,5-Bis(4-tert-butylphenyl)thiophen-2-yl)-1H-tetrazole (DNM0546)

¹H NMR (DMSO, 500 MHz) δ 7.84 (s, 1H), 7.53-7.37 (m, 4H), 7.30 (d,J=8.41 Hz, 2H), 7.27 (d, J=8.41 Hz, 2H), 1.29 (s, 9H), 1.28 (s, 9H); ¹³CNMR (DMSO, 125 MHz) 151.15, 150.07, 141.08, 132.17, 132.12, 129.84,128.46, 128.28, 125.80, 125.53, 34.46, 34.37, 31.09, 30.99.

5-(4,5-Bis(4-chloro-2-iso-propylphenyl)thiophen-2-yl)-1H-tetrazole(DNM0548)

¹H NMR (DMSO, 500 MHz) δ 7.75 (s, 1H), 7.43-7.39 (m, 2H), 7.31-7.26 (m,2H), 7.30 (dd, J=2.24 Hz, J₂=8.30 Hz, 1H), 7.06 (d, J=8.30 Hz, 1H),2.97-2.82 (m, 2H), 1.04 (broad, 6H), 0.95 (d, J=6.80 Hz, 6H); ¹³C NMR(DMSO, 125 MHz) δ 150.00, 149.20, 140.39, 138.88, 134.47, 133.44,133.30, 132.20, 131.71, 131.19, 128.76, 126.16, 126.04, 125.97, 125.76,29.95, 29.77, 23.54 (broad).

5-(4,5-Bis(2-(trifluoromethyl)phenyl)thiophen-2-yl)-1,1-tetrazole(DNM0549)

¹H NMR (DMSO, 500 MHz) δ 7.88-7.78 (m, 3H), 7.66-7.58 (m, 2H), 7.56-7.49(m, 2H), 7.41 (broad, 1H), 7.23-7.15 (m, 1H); ¹³C NMR (DMSO, 125 MHz) δ

5-(4,5-Bis(2-(trifluoromethoxy)phenyl)thiophen-2-yl)-1H-tetrazole(DNM0550)

¹H NMR (DMSO, 500 MHz) δ 7.90 (s, 1H), 7.57-7.51 (m, 1H), 7.51-7.46 (m,1H), 7.45-7.34 (m, 5H), 7.32 (dd, J₁=7.50 Hz, J₂=1.70 Hz, 1H); ¹³C NMR(DMSO, 125 MHz) δ 145.44, 136.94, 135.8, 132.48, 131.7, 131.17, 130.98,130.22, 127.82, 127.62, 127.4, 125.37, 120.88, 120.54, 118.85, 118.81.

5-(4,5-Bis(2,4-dimethoxyphenyl)thiophen-2-yl)-1H-tetrazole (DNM0552)

¹H NMR (DMSO, 500 MHz) δ 7.75 (s, 1H), 6.99 (d, J=8.45 Hz, 1H), 6.87 (d,J=8.45 Hz, 1H), 6.64-6.58 (m, 2H), 6.46 (dd, J₁=8.50 Hz, J₂=2.25 Hz,1H), 6.43 (dd, J₁=8.50 Hz, J₂=2.25 Hz, 1H), 3.764 (s, 3H), 3.756 (s,3H), 3.66 (s, 3H), 3.65 (s, 3H); ¹³C NMR (DMSO, 125 MHz) δ 160.84,160.13, 157.52, 157.46, 138.46, 136.36, 131.91, 131.62, 130.91, 117.24,114.29, 105.25, 104.89, 98.82, 98.79, 55.49, 55.40, 55.29, 55.19.

5-(4,5-Bis(2,6-dimethoxyphenyl)thiophen-2-yl)-1H-tetrazole (DNM0553)

¹H NMR (DMSO, 500 MHz) δ 7.68 (s, 1H), 7.22 (t, J=8.38 Hz, 1H), 7.16 (d,J=8.33 Hz, 1H), 6.57 (d, J=8.40 Hz, 2H), 6.55 (d, J=8.35 Hz, 1H), 3.49(s, 6H), 3.47 (s, 6H); ¹³C NMR (DMSO, 125 MHz) δ 157.67, 157.29, 134.19,132.23, 130.01, 128.89, 113.22, 110.53, 103.80, 103.73, 55.26, 55.10.

(2,2′-(5-(1H-tetrazol-5-yl)thiophene-2,3-diyl)bis(2,1-phenylene))dimethanol(DNM0555)

¹H NMR (DMSO, 500 MHz) δ 7.85 (s, 1H), 7.51 (d, J=7.60 Hz, 1H), 7.48 (d,J=7.70 Hz, 1H), 7.37 (dt, J₁=1.47 Hz, J₂=7.45 Hz, 1H), 7.33-7.21 (m,3H), 7.13 (dt, J₁=1.00 Hz, J₂=7.49 Hz, 1H), 7.02 (dd, J₁=1.00 Hz,J₂=7.60 Hz, 1H), 4.36 (s, 2H), 4.23 (s, 2H); ¹³C NMR (DMSO, 125 MHz) δ141.19, 140.84, 140.12, 139.48, 133.01, 131.45, 131.19, 130.04, 129.89,128.87, 127.85, 127.65, 127.26, 126.65, 126.59, 60.71, 60.44.

2,2′-(5-(1H-tetrazol-5-yl)thiophene-2,3-diyl)dibenzaldehyde (DNM0556)

¹H NMR (CDCl₃, 500 MHz) δ 9.91 (s, 1H), 9.80 (s, 1H), 7.93 (s, 1H), 7.75(d, J=7.80 Hz, 2H), 7.59 (dt, J₁=1.35 Hz, J₂=7.55 Hz, 1H), 7.49-7.41 (m,3H), 7.34 (t, J=7.53 Hz, 1H), 7.18 (d, J=7.50 Hz, 1H); ¹³C NMR (CDCl₃,125 MHz) δ 192.30, 191.20, 153.22, 140.91, 139.47, 137.51, 134.79,134.72, 134.66, 134.40, 134.04, 133.00, 132.44, 132.21, 130.28, 129.89,129.76, 129.32, 126.53.

5-(4,5-Di(furan-3-yl)thiophen-2-yl)-1H-tetrazole (DNM0557)

¹H NMR (DMSO, 500 MHz) δ 8.03 (dd, J₁=0.93 Hz, J₂=1.38 Hz, 1H), 7.93(dd, J₁=0.90 Hz, J₂=1.30 Hz, 1H), 7.87 (s, 1H), 7.83 (t, J=1.60 Hz, 1H),7.77 (t, J=1.70 Hz, 1H), 6.63 (dd, J₁=0.83 Hz, J₂=1.83 Hz, 1H), 6.59(dd, J₁=0.80 Hz, J₂=1.80 Hz, 1H); ¹³C NMR (DMSO, 125 MHz) δ 144.46,143.86, 141.52, 140.76, 131.73, 130.93, 130.30, 119.66, 117.66, 110.91,110.38.

5-(4,5-Di(thiophen-3-yl)thiophen-2-yl)-1H-tetrazole (DNM0558)

¹H NMR (DMSO, 500 MHz) δ 7.89 (s, 1H), 7.68 (dd, J₁=1.33 Hz, J₂=2.88 Hz,1H), 7.65 (dd, J₁=2.95 Hz, J₂=4.95 Hz, 1H), 7.61 (dd, J₁=2.95 Hz,J₂=4.90 Hz, 1H), 7.59 (dd, J₁=1.28 Hz, J₂=2.88 Hz, 1H), 7.03 (dd,J₁=1.18 Hz, J₂=4.93 Hz, 1H); ¹³C NMR (DMSO, 125 MHz) δ 135.88, 135.23,133.66, 132.73, 131.40, 127.61, 127.59, 127.52, 126.80, 125.14, 123.88.

5-(4,5-Bis(2-(thiophen-3-yl)phenyl)thiophen-2-yl)-1H-tetrazole (DNM0559)

¹H NMR (DMSO, 500 MHz) δ 7.65 (s, 1H), 7.40-7.35 (m, 2H), 7.32 (dt,J₁=1.15 Hz, J₂=7.51 Hz, 1H), 7.29-7.23 (m, 3H), 7.16-7.00 (m, 2H), 6.90(dd, J₁=1.23 Hz, J₂=2.88 Hz, 1H), 6.85 (dd, J₁=1.23 Hz, J₂=2.88 Hz, 1H),6.73 (d, J=7.65 Hz, 1H), 6.64 (d, J=7.65 Hz, 1H), 6.58 (dd, J₁=1.20 Hz,J₂=4.95 Hz, 1H), 6.53 (dd, J₁=1.20 Hz, J₂=4.95 Hz, 1H); ¹³C NMR (DMSO,125 MHz) δ 142.01, 140.99, 140.62, 139.74, 135.76, 135.02, 132.86,131.72, 130.96, 130.16, 129.91, 129.73, 129.37, 128.68, 127.94, 127.81,127.73, 127.16, 127.06, 125.56, 125.39, 123.16, 122.78.

5-(4,5-Bis(2-(furan-3-yl)phenyl)thiophen-2-yl)-1H-tetrazole (DNM0560)

¹H NMR (DMSO, 500 MHz) δ 7.61 (s, 1H), 7.59-7.56 (m, 2H), 7.38-7.31 (m,3H), 7.30-7.25 (m, 2H), 7.25-7.22 (m, 1H), 7.19-7.14 (m, 1H), 7.12 (dt,J₁=1.30 Hz, J₂=7.58 Hz, 1H), 6.96 (d, J=7.54 Hz, 1H), 6.85 (dd, J=0.95Hz, J₂=7.66 Hz, 1H), 6.20 (dd, J₁=0.83 Hz, J₂=1.78 Hz, 1H), 6.12 (dd,J₁=0.80 Hz, J₂=1.75 Hz, 1H); ¹³C NMR (DMSO, 125 MHz) δ 142.01, 140.99,140.62, 139.74, 135.76, 135.02, 132.86, 131.72, 130.96, 130.16, 129.91,129.73, 129.37, 128.68, 127.94, 127.81, 127.73, 127.16, 127.06, 125.56,125.39, 123.16, 122.78.

5-(4,5-Bis(2-chloro-4-methylphenyl)thiophen-2-yl)-1H-tetrazole (DNM0563)

¹H NMR (DMSO, 500 MHz) δ 7.85 (s, 1H), 7.36-7.32 (m, 2H), 7.29 (d,J=7.80 Hz, 1H), 7.06 (d, J=7.74 Hz, 1H), 7.09-7.02 (m, 2H), 2.30 (s,3H), 2.28 (s, 3H); ¹³C NMR (DMSO, 125 MHz) δ 141.00, 139.73, 138.42,132.59, 132.54, 131.88, 131.38, 131.08, 130.57, 130.19, 130.03, 128.06,127.87, 127.82, 20.39, 10.37.

5-(4,5-Bis(4-methoxy-3,5-dimethylphenyl)thiophen-2-yl)-1H-tetrazole(DNM0564)

¹H NMR (DMSO, 500 MHz) δ 7.83 (s, 1H), 7.03 (s, 2H), 6.99 (s, 2H), 3.67(s, 6H), 2.172 (s, 6H), 2.166 (s, 6H); ¹³C NMR (DMSO, 125 MHz) δ 156.89,156.12, 140.69, 137.89, 131.78, 130.86, 130.45, 130.27, 129.19, 128.99,127.91, 59.43, 59.42, 15.78, 15.74.

5-(4,5-Bis(3-chloro-4-methoxyphenyl)thiophen-2-yl)-1H-tetrazole(DNM0565)

¹H NMR (DMSO, 500 MHz) δ 7.87 (s, 1H), 7.41 (d, J=2.05 Hz, 1H), 7.39 (d,J=2.00 Hz, 1H), 7.30 (dd, J₁=2.15 Hz, J₂=8.65 Hz, 1H), 7.22 (dd, J₁=2.08Hz, J₂=8.58 Hz, 1H), 7.20 (d, J=8.70 Hz, 1H), 7.17 (d, J=8.65 Hz, 1H),3.89 (s, 3H), 3.88 (s, 3H); NMR (DMSO, 125 MHz) δ 172.03, 154.75,154.03, 139.42, 136.95, 131.63, 130.00, 129.84, 129.19, 128.70, 127.88,125.48, 121.39, 121.18, 113.24, 113.01, 56.29, 56.18.

Diethyl 3,3′-(5-(1H-tetrazol-5-yl)thiophene-2,3-diyl)dibenzoate(DNM0593)

¹H NMR (CDCl₃, 500 MHz) δ 8.17 (s, 1H), 8.14 (s, 1H), 8.06 (s, 1H), 8.03(d, J=7.75 Hz, 1H), 7.98 (d, J=7.75 Hz, 1H), 7.48 (d, J=7.90 Hz, 1H),7.44-7.37 (m, 3H), 7.34 (t, J=7.75 Hz, 1H), 4.44-4.35 (m, 4H), 1.42-1.35(m, 6H); ¹³C NMR (CDCl₃, 125 MHz) δ 167.33, 166.35, 142.50, 138.70,135.47, 133.75, 133.73, 133.17, 132.49, 131.28, 131.27, 130.90, 130.44,130.20, 129.85, 129.20, 128.99, 128.97, 124.48, 61.88, 61.64, 14.39.

1,1′-(3,3′-(5-(1H-tetrazol-5-yl)thiophene-2,3-diyl)bis(3,1-phenylene))dibutan-1-one(DNM0599)

¹H NMR (CDCl₃, 500 MHz) δ 8.13 (s, 1H), 8.08 (s, 1H), 7.98-7.89 (m, 3H),7.54 (d, J=7.80 Hz, 1H), 7.46 (t, J=7.60 Hz, 1H), 7.40 (t, J=7.70 Hz,1H), 2.94 (t, J=7.28 Hz, 2H), 2.85 (t, J=7.30 Hz, 2H), 1.80-1.67 (m,4H), 0.99 (t, J=7.35 Hz, 3H), 0.98 (t, J=7.35 Hz, 3H); ¹³C NMR (CDCl₃,125 MHz) δ 200.52, 139.04, 137.96, 137.80, 136.06, 134.09, 134.00,133.71, 132.47, 129.80, 129.51, 129.44, 129.04, 128.58, 128.04, 41.20,41.06, 18.23, 18.18, 14.28.

Diethyl5,5′-(5-(1H-tetrazol-5-yl)thiophene-2,3-diyl)bis(3-hydroxybenzoate)(DNM0607)

¹H NMR (DMSO, 500 MHz) δ 10.12 (s, 1H), 9.99 (s, 1H), 7.87 (s, 1H),7.43-7.29 (m, 4H), 6.97 (s, 1H), 6.93 (s, 1H), 4.35-4.17 (m, 4H),1.33-1.20 (m, 6H); ¹³C NMR (DMSO, 125 MHz) δ 165.34, 165.07, 157.89,157.75, 139.98, 138.09, 136.36, 133.97, 131.78, 131.60, 131.02, 120.17,120.10, 120.01, 115.86, 115.11, 60.91, 60.81, 14.07, 14.00.

5-(4,5-Bis(3-butylphenyl)thiophen-2-yl)-1H-tetrazole (DNM0608)

¹H NMR (CDCl₃, 500 MHz) δ 7.44 (s, 1H), 6.91-6.56 (m, 8H), 8.06 (s, 1H),2.31-2.05 (m, 4H), 1.29-1.16 (m, 4H), 1.16-1.00 (m, 4H), 0.81-0.67 (m,6H); ¹³C NMR (CDCl₃, 125 MHz) δ 142.80, 142.62, 139.91, 138.91, 136.26,134.07, 129.86, 129.58, 129.54, 128.54, 127.63, 127.02, 126.88, 126.61,35.70, 35.68, 33.79, 33.68, 22.66, 22.63, 14.33.

5-(4,5-bis(3-(cyclopentylmethyl)phenyl)thiophen-2-yl)-1H-tetrazole(DNM0612)

¹H NMR (CDCl₃, 500 MHz) δ 8.00 (s, 1H), 7.24-7.17 (m, 2H), 7.17-7.09 (m,4H), 7.09-7.04 (m, 2H), 2.54-2.48 (m, 4H), 2.00-1.87 (m, 2H), 1.69-1.54(m, 8H), 1.54-1.41 (m, 4H), 1.17-1.00 (m, 4H); ¹³C NMR (CDCl₃, 125 MHz)δ 152.84, 144.09, 143.03, 142.82, 139.94, 135.22, 133.02, 132.94,129.97, 129.77, 129.12, 128.70, 128.57, 128.14, 126.76, 126.41, 122.42,42.08, 41.99, 32.56, 25.06.

Example 2 Synthesis of DNM0576 and Analogues

4-Bromo-5-(4-methylnaphthalen-1-yl)thiophene-2-carbonitrile

A round-bottomed flask was charged with4,5-dibromothiophene-2-carbonitrile (536 mg, 2.00 mmol),4-methylnaphthalen-1-ylboronic acid (409 mg, 2.20 mmol), Pd₂(dba)₃ (18.3mg, 0.020 mmol) and KF (383 mg, 6.60). After degassed, dioxane (5 mL)and P(Bu-t)₃ (0.24 mL, 0.2M, 0.048 mmol) was added. The reaction mixturewas stirred at rt until complete. 30 mL of water was added, and thereaction mixture was extracted with ethyl acetate. The organic phase wasdried over anhydrous sodium sulphate and then concentrated. The residuewas purified by flash chromatography (hexane:CH₂Cl₂=3:1). 0.58 g (88%)of product was obtained as a white solid.

4-(4-Chloro-2-methylphenyl)-5-(4-methylnaphthalen-1-yl)thiophene-2-carbonitrile

A round-bottomed flask was charged with4-bromo-5-(4-methylnaphthalen-1-yl)thiophene-2-carbonitrile (203 mg,0.62 mmol), 4-chloro-2-methylphenylboronic acid (119 mg, 0.70 mmol),Pd₂(dba)₃ (9.2 mg, 0.010 mmol) and KF (126 mg, 2.17). After degassed,dioxane (2.0 mL) and P(Bu-t)₃ (0.15 mL, 0.2M, 0.03 mmol) was added. Thereaction mixture was stirred at rt until complete. 20 mL of water wasadded, and the reaction mixture was extracted with ethyl acetate. Theorganic phase was dried over anhydrous sodium sulphate and thenconcentrated. The residue was purified by flash chromatography(hexane:CH₂Cl₂=3:1). 0.21 g (90.6%) of product was obtained as a whitesolid.

5-(4-(4-Chloro-2-methylphenyl)-5-(4-methylnaphthalen-1-yl)thiophen-2-yl)-1H-tetrazole(DNM0576)

A round-bottomed flask was charged with4-(4-chloro-2-methylphenyl)-5-(4-methylnaphthalen-1-yl)thiophene-2-carbonitrile(209 mg, 0.56 mmol), zinc bromide (338 mg, 1.50 mmol) and sodium azide(97.5 mg, 1.50 mmol). After degassed, DMF (3 mL) was added. The reactionmixture was heated to 110° C. and stirred at this temperature untilcomplete. The reaction was cooled to rt, and 30 mL of 0.1 N aqueous HClwas added. The reaction mixture was extracted with ethyl acetate. Theorganic phase was dried over anhydrous sodium sulphate and thenconcentrated. The residue was purified by flash chromatography(hexane:EtOAc:AcOH=30:10:1). 196 mg (84%) of product was obtained as awhite solid, ¹H NMR (DMSO, 500 MHz) δ 8.03 (d, J=8.40 Hz, 1H), 7.88 (s,1H), 7.80 (d, J=8.05 Hz, 1H), 7.55 (t, J=7.68 Hz, 1H), 7.48 (t, J=7.65Hz, 1H), 7.45 (d, J=7.19 Hz, 1H), 7.37 (d, J=7.30 Hz, 1H), 7.23 (d,J=1.95 Hz, 1H), 7.09 (d, J=8.25 Hz, 1H), 7.04 (dd, J=8.25 Hz, J₂=2.06Hz, 1H), 2.65 (s, 3H), 2.17 (s, 3H); ¹³C NMR (DMSO, 125 MHz) δ 172.05,141.01, 139.65, 138.35, 135.93, 133.73, 132.18, 132.10, 131.64, 131.31,131.26, 129.80, 129.23, 127.62, 126.45, 126.26, 126.02, 125.53, 125.51,124.67, 21.08, 19.82.

The following compounds were also prepared using an analogous method:

5-(4-(4-Chlorophenyl)-5-(4-methylnaphthalen-1-yl)thiophen-2-yl)-1H-tetrazole(DNM0572)

¹H NMR (CDCl₃, 500 MHz) δ 8.09 (d, J=8.39 Hz, 1H), 7.84 (s, 1H), 7.82(d, J=8.46 Hz, 1H), 7.56 (t, J=7.32 Hz, 1H), 7.44 (t, J=7.84 Hz, 1H),7.31 (d, J=7.25 Hz, 1H), 7.23 (d, J=7.24 Hz, 1H), 7.17-7.09 (m, 4H),2.76 (s, 3H); ¹³C NMR (CDCl₃, 125 MHz) δ 152.64, 142.65, 138.24, 135.00,134.09, 133.65, 133.08, 132.16, 131.91, 131.87, 129.76, 128.93, 127.95,126.54, 126.37, 126.30, 126.11, 124.78, 124.46, 19.80.

5-(4-(5-Chlorophenyl)-4-(4-methylnaphthalen-1-yl)thiophen-2-yl)-1H-tetrazole(DNM0575)

¹H NMR (DMSO, 500 MHz) δ 8.11 (s, 1H), 8.09 (d, J=8.44 Hz, 1H), 7.65 (d,J=8.11 Hz, 1H), 7.58-7.53 (m, 2H), 7.49-7.42 (m, 2H), 7.28-7.20 (m, 4H),2.72 (s, 3H); ¹³C NMR (DMSO, 125 MHz) δ 139.74, 139.15, 136.18, 133.66,132.29, 132.15, 131.14, 130.40, 129.40, 129.23, 128.61, 127.69, 126.69,126.36, 126.28, 125.47, 124.77, 19.23.

5-(4-(4-Fluorophenyl)-5-(4-(methylsulfonyl)phenyl)thiophen-2-yl)₄H-tetrazole(DNM0592)

¹H NMR (DMSO, 500 MHz) δ 7.96 (s, 1H), 7.94 (d, J=8.45 Hz, 2H), 7.57 (d,J=8.45 Hz, 2H), 7.45-7.38 (m, 2H), 7.28 (t, J=8.80 Hz, 2H), 3.27 (s,31-1); ¹³C NMR (DMSO, 125 Hz) δ 163.32, 161.36, 141.61, 139.83, 139.71,137.14, 131.45, 131.38, 131.32, 129.58, 129.42, 128.44, 128.42, 127.46,124.88, 116.37, 116.19, 43.36.

Ethyl3-(2-(4-methylnaphthalen-1-yl)-5-(1H-tetrazol-5-yl)thiophen-3-yl)benzoate(DNM0596)

¹H NMR (CDCl₃, 500 MHz) δ 8.00 (d, J=8.40 Hz, 1H), 7.96 (t, J=1.64 Hz,1H), 7.87 (s, 1H), 7.83 (dt, J₁=7.85 Hz, J₂=1.35 Hz, 1H), 7.73 (d,J=8.36 Hz, 1H), 7.35 (t, J=7.60 Hz, 1H), 7.28 (d, J=7.79 Hz, 1H), 7.22(d, J=7.74 Hz, 1H), 7.17 (d, J=7.10 Hz, 1H), 7.14 (t, J=7.85 Hz, 1H),4.24 (q, J=7.07 Hz, 2H), 2.68 (s, 3H), 1.24 (t, J=7.15 Hz, 3H); ¹³C NMR(CDCl₃, 125 MHz) δ166.77, 143.43, 138.88, 135.13, 134.17, 133.51,133.05, 132.84, 132.03, 131.41, 130.72, 129.59, 129.31, 128.91, 127.95,126.46, 126.36, 126.14, 124.71, 123.55, 61.60, 19.76, 14.21.

Ethyl3-(2-(4-chloro-2-methylphenyl)-5-(1H-tetrazol-5-yl)thiophen-3-yl)benzoate(DNM0597)

¹H NMR (CDCl₃, 500 MHz) δ 8.01-7.95 (m, 2H), 7.78 (s, 1H), 7.41-7.33 (m,2H), 7.23 (s, 1H), 7.19 (dd, J₁=8.22 Hz, J₂=1.50 Hz, 1H), 7.14 (d,J=8.22 Hz, 1H), 4.38 (q, J=7.15 Hz, 2H), 2.04 (s, 3H), 1.40 (t, J=7.14Hz, 3H); ¹³C NMR (CDCl₃, 125 MHz) δ166.58, 142.77, 138.62, 138.53,134.15, 133.77, 133.52, 132.76, 132.50, 131.59, 131.14, 130.70, 129.59,129.35, 129.24, 126.57, 124.34, 61.74, 20.16, 14.39.

Example 3 Synthesis of DNM0574 and Analogues

4,5-Dibromothiazole

To a solution of 2,4,5-triibromothiazole (3.12 g, 9.67 mmol) inanhydrous THF (25 mL) was added i-PrMgCl (4.84 mL, 2M in THF, 9.67 mmol)at ice-salt bath temperature under argon. After complete addition, thereaction was stirred for 1 h at ice-salt bath temperature, and thenquenched with methanol (2 mL). The reaction was worked up with a typicalprocedure, and the crude product was purified by flash chromatography(30% of DCM in hexane) to afford 1.45 g of product.

4,5-Bis(4-chloro-2-methylphenyl)thiazole

A round-bottomed flask was charged with 4,5-dibromothiazole (160 mg,0.66 mmol), 4-chloro-2-methylphenylboronic acid (269 mg, 1.58 mmol) andPd(PPh₃)₄ (38.1 mg, 0.033 mmol). After degassed, dioxane (5 mL) andaqueous sodium carbonate (3 mL, 2M, 6 mmol) was added. The reactionmixture was heated to 90° C. The progress of the reaction was monitoredby TLC. After the reaction was complete, 50 mL of water was added, andthe reaction mixture was extracted with ethyl acetate. The organic phasewas dried over anhydrous sodium sulphate and then concentrated. Theresidue was purified by flash chromatography to afford 170 mg of productas clear oil.

2-Bromo-4,5-bis(4-chloro-2-methylphenyl)thiazole

A solution of 4,5-Bis(4-chloro-2-methylphenyl)thiazole (170 mg, 0.51mmol), NBS (100 mg, 0.56 mmol) and NaOAc (82 mg, 1.0 mmol) in AcOH (5mL) and DCM (3 mL) was stirred overnight at rt, and then quenched withwater (25 mL) and extracted with ethyl acetate. The organic phase wasdried over anhydrous sodium sulphate and then concentrated. The residuewas purified by flash chromatography to afford 196 mg of product.

4,5-Bis(4-chloro-2-methylphenyl)thiazole-2-carbonitrile

A round-bottomed flask was charged with2-Bromo-4,5-bis(4-chloro-2-methylphenyl)thiazole (196 mg, 0.47 mmol) andCuCN (84.2 mg, 0.94 mmol). After degassed, 3 mL of DMF was added. Thereaction mixture was heated to 150° C. overnight. After cooled to roomtemperature, the reaction was quenched with 25 mL of water, andextracted with ethyl acetate. The organic phase was dried over anhydroussodium sulphate and then concentrated. The residue was purified by flashchromatography to afford 150 mg of product.

4,5-Bis(4-chloro-2-methylphenyl)-2-(1H-tetrazol-5-yl)thiazole (DNM0574)

A round-bottomed flask was charged with4,5-bis(4-chloro-2-methylphenyl)thiazole-2-carbonitrile (150 mg, 0.42mmol), zinc bromide (189 mg, 0.84 mmol) and sodium azide (45.5 mg, 0.84mmol). After degassed, DMF (3 mL) was added. The reaction mixture washeated to 150° C. overnight. After cooled to rt, the reaction wasquenched with 30 mL of 0.1 N aqueous HCl, and extracted with ethylacetate. The organic phase was dried over anhydrous sodium sulphate andthen concentrated. The residue was purified by flash chromatography(hexane:EtOAc:AcOH=30:10:1). 120 mg of product was obtained as a whitesolid, ¹H NMR (DMSO, 500 MHz) δ 7.93 (d, J=8.41 Hz, 1H), 7.57 (d, J=1.90Hz, 1H), 7.49-7.44 (m, 2H), 7.33-7.28 (m, 2H), 2.63 (s, 3H), 2.18 (s,3H); ¹³C NMR (DMSO, 125 MHz) δ 166.07, 153.61, 139.50, 138.74, 134.92,133.48, 132.53, 131.87, 131.42, 130.27, 129.98, 126.64, 125.67, 21.13,19.51.

The following compounds were also prepared using an analogous method:

4,5-Bis(5-chloro-2-methylphenyl)-2-(1H-tetrazol-5-yl)thiazole (DNM0567)

¹H NMR (DMSO, 500 MHz) δ 7.95 (d, J=2.17 Hz, 1H), 7.54 (dd, J₁=8.25 Hz,J₂=1.80 Hz, 1H), 7.48 (d, J=8.25 Hz, 1H), 7.45 (dd, J₁=8.23 Hz, J₂=2.33Hz, 1H), 7.41-7.36 (m, 2H), 2.61 (s, 3H), 2.11 (s, 3H); ¹³C NMR (DMSO,125 MHz) δ 165.31, 152.99, 135.93, 135.54, 135.31, 133.72, 133.07,132.04, 130.99, 130.09, 129.94, 129.64, 128.79, 128.72, 20.82, 19.03.

4,5-Bis(3-chloro-4-methylphenyl)-2-(1H-tetrazol-5-yl)thiazole (DNM0568)

¹H NMR (DMSO, 500 MHz) δ 8.09 (d, J=1.30 Hz, 1H), 7.92 (dd, J₁=7.93 Hz,J₂=1.38 Hz, 1H), 7.79 (d, J=1.20 Hz, 1H), 7.57-7.50 (m, 2H), 7.43 (d,J=7.95 Hz, 1H), 2.42 (s, 3H), 2.39 (s, 3H); ¹³C NMR (DMSO, 125 MHz) δ166.30, 153.18, 138.95, 136.51, 134.29, 133.22, 132.68, 132.20, 131.49,131.20, 129.01, 127.53, 126.22, 125.36, 19.68, 19.50.

4,5-Bis(4-methylnaphthalen-1-yl)-2-(1H-tetrazol-5-yl)thiazole (DNM0569)

¹H NMR (DMSO, 500 MHz) δ 9.99 (d, J=7.91 Hz, 1H), 8.16 (t, J=9.52 Hz,2H), 8.04 (d, J=7.35 Hz, 1H), 7.86 (d, J=8.41 Hz, 1H), 7.47-7.49 (m,6H), 7.47 (d, J=7.30 Hz, 1H), 2.764 (s, 3H), 2.761 (s, 3H); ¹³C NMR(DMSO, 125 MHz) δ 167.71, 154.55, 138.21, 135.74, 132.61, 132.41,131.55, 129.79, 129.58, 129.06, 128.26, 127.48, 127.41, 126.69, 126.34,126.23, 126.13, 126.04, 125.93, 125.83, 124.78, 124.59, 19.52, 19.23.

4,5-Bis(4-chlorophenyl)-2-(1H-tetrazol-5-yl)thiazole (DNM0573)

¹H NMR (DMSO, 500 MHz) δ 8.10 (d, J=8.43 Hz, 2H), 7.74 (d, J=8.44 Hz,2H), 7.64 (d, J=7.50 Hz, 2H), 53 (d, J=8.50 Hz, 2H); ¹³C NMR (DMSO, 125MHz) δ 166.61, 153.57, 135.94, 133.87, 132.11, 130.85, 130.74, 129.52,128.50, 128.29.

4,5-Bis(4-fluoronaphthalen-1-yl)-2-(1H-tetrazol-5-yl)thiazole (DNM0578)

¹H NMR (DMSO, 500 MHz) δ 9.02-8.95 (m, 1H), 8.24-8.16 (m, 3H), 7.88 (d,J=8.49 Hz, 1H), 7.79-7.73 (m, 1H), 7.73-7.67 (m, 2H), 7.62 (t, J=7.80Hz, 1H), 7.58 (dd, J₁=8.21 Hz, J₂=10.14 Hz, 1H), 7.47 (dd, J₁=7.96 Hz,J₂=10.54 Hz, 1H); ¹³C NMR (DMSO, 125 MHz) δ 166.76, 160.47, 159.60,158.43, 157.59, 153.30, 132.99, 132.96, 131.08, 130.07, 129.99, 129.08,128.99, 128.92, 127.82, 127.58, 126.92, 125.63, 123.39, 123.26, 123.23,123.10, 120.54, 120.49, 120.35, 120.30, 109.92, 109.75, 109.55, 109.40.

4,5-Bis(4-biphenyl)-2-(1H-tetrazol-5-yl)thiazole (DNM0581)

¹H NMR (DMSO, 500 MHz) δ 8.17 (d, J=8.32 Hz, 2H), 7.89 (d, J=8.32 Hz,2H), 7.84-7.72 (m, 8H), 7.56-7.47 (m, 4H), 7.47-7.38 (m, 2H); ¹³C NMR(DMSO, 125 MHz) δ 167.64, 154.70, 142.75, 140.67, 139.37, 138.93,132.38, 131.10, 129.43, 129.12, 129.05, 128.23, 127.84, 127.60, 127.17,126.79, 126.70, 126.68.

4,5-Bis(4-t-butylphenyl)-2-(1H-tetrazol-5-yl)thiazole (DNM0582)

¹H NMR (DMSO, 500 MHz) δ 7.99 (d, J=8.25 Hz, 2H), 7.60 (d, J=8.25 Hz,4H), 7.48 (d, J=8.30 Hz, 2H), 1.34 (s, 9H), 1.32 (s, 9H); ¹³C NMR (DMSO,125 MHz) δ 167.93, 155.03, 154.21, 151.65, 130.58, 129.56, 128.52,126.35, 126.27, 125.27, 34.77, 34.51, 31.02, 30.87.

4,5-Bis(3,4-dimethylphenyl)-2-(1H-tetrazol-5-yl)thiazole (DNM0583)

¹H NMR (DMSO, 500 MHz) δ 7.85 (s, 1H), 7.79 (dd, J₁=1.65, J₂=7.80 Hz,1H), 7.47 (s, 1H), 7.34 (d, J=7.95 Hz, 1H), 7.29 (d, J=7.70 Hz, 1H),7.19 (d, J=7.85 Hz, 1H), 2.34 (s, 3H), 2.31 (s, 3H), 2.28 (s, 3H), 2.25(s, 3H); ¹³C NMR (DMSO, 125 MHz) δ 168.65, 155.73, 140.64, 138.00,137.85, 136.73, 131.33, 130.85, 130.32, 130.17, 129.93, 127.70, 126.54,124.49, 19.91, 19.87, 19.75, 19.71.

4,5-Bis(4-chloro-2-isopropylphenyl)-2-(1H-tetrazol-5-yl)thiazole(DNM0584)

¹H NMR (DMSO, 500 MHz) δ 7.73 (d, J=8.35 Hz, 1H), 7.62 (d, J=2.20 Hz,1H), 7.52 (d, J=2.10 Hz, 1H), 7.47 (dd, J₁=2.18, J₂=8.33 Hz, 1H), 7.30(dd, J₁=2.13, J₂=8.23 Hz, 1H), 7.25 (d, J=8.20 Hz, 1H), 3.77 (sep,J=6.85 Hz, 1H), 2.93 (sep, J=6.83 Hz, 1H), 1.21 (d, J=6.85 Hz, 6H), 1.11(d, J=6.80 Hz, 6H); ¹³C NMR (DMSO, 125 MHz) δ 166.64, 154.25, 150.51,150.06, 136.15, 134.62, 132.72, 132.50, 131.94, 129.84, 127.13, 126.93,126.22, 126.16, 30.57, 29.36, 23.72, 23.59.

Example 4 Synthesis of DNM0577 and Analogues

4,5-Diiodothiophene-2-carboxamide

Thiophene-2-carbonitrile (5.39 g, 49.39 mmol) was dissolved in a mixtureof H₂SO₄ (10 mL) and TFA (30 mL). NIS (23.34 g, 103.74 mmol) was addedin portion in 30 min. After complete addition, the reaction mixture wasfurther stirred for 4 h, and then poured onto 300 g of crushed ice. Awhite solid was formed, which was collected, washed with water, anddried together with phosphorous pentoxide under vacuum to afford 17.8 g(95%) of product.

4,5-Diiodothiophene-2-carbonitrile

An oven-dried round-bottomed flask was charged with4,5-diiodothiophene-2-carboxamide (5.68 g, 15.0 mmol) and 20 mL of DMFunder an argon atmosphere. The solution was cooled on an ice-water bath,and cyanuric chloride (1.81 g, 9.8 mmol) was then added in one portion.After stirring at 0° C. for 1 h, the reaction mixture was warmed to roomtemperature, and stirred for a further 3 h. 100 mL of water was added. Awhite solid was formed, which was collected through suction filtration,washed with water, and dried together with phosphorous pentoxide undervacuum to afford 4.87 g (90%) of product.

5-((4-Chlorophenyl)(hydroxy)methyl)-4-iodothiophene-2-carbonitrile

An oven-dried round-bottomed flask was charged with4,5-diiodothiophene-2-carbonitrile (1.80 g, 5.0 mmol). After degassed,anhydrous THF (15 mL) was added through syringe. After cooled to −78°C., i-PrMgCl (3 mL, 2.0 M, 6.0 mmol) was added dropwise. The reactionwas further stirred for 30 min at −78° C. after complete addition.4-chlorobenzaldehyde (0.98 g, 7.0 mmol) in 5 mL THF was then added.After stirring for 10 min at −78° C. for 1 h, the reaction mixture waswarmed to room temperature. 50 mL of saturated aqueous NH₄Cl was added.The reaction mixture was extracted with ethyl acetate. The organic phasewas dried over anhydrous sodium sulphate and then concentrated. Theresidue was purified by flash chromatography(hexane:EtOAc:CH₂CL₂=30:3:10). 1.39 g (74%) of product was obtained as awhite solid.

5-(4-Chlorobenzyl)-4-iodothiophene-2-carbonitrile

A round-bottomed flask was charged with5-((4-chlorophenyl)(hydroxy)methyl)-4-iodothiophene-2-carbonitrile (1.67g, 4.45 mmol). After degassed, dichloromethane (8 mL), TFA (4 mL) andtriethylsilane (1.1 mL, 6.9 mmol) was added sequentially throughsyringe. The reaction was further stirred at rt until complete, and thenconcentrated. The residue was purified by flash chromatography(hexane:CH₂CL₂=2:1). 1.36 g (85%) of product was obtained as a whitesolid.

5-(4-Chlorobenzyl)-4-(4-methylnaphthalen-1-yl)thiophene-2-carbonitrile

A round-bottomed flask was charged with5-(4-chlorobenzyl)-4-iodothiophene-2-carbonitrile (180 mg, 0.50 mmol),4-methylnaphthalen-1-ylboronic acid (102 mg, 0.55 mmol), Pd₂(dba)₃ (9.2mg, 0.010 mmol) and KF (126 mg, 2.17). After degassed, dioxane (2.0 mL)and P(Bu-t)₃ (0.15 mL, 0.2M, 0.03 mmol) was added. The reaction mixturewas stirred at rt until complete. 20 mL of water was added, and thereaction mixture was extracted with ethyl acetate. The organic phase wasdried over anhydrous sodium sulphate and then concentrated. The residuewas purified by flash chromatography (5% ethyl acetate in hexane). 0.17g (91%) of product was obtained as a white solid.

5-(5-(4-Chlorobenzyl)-4-(4-methylnaphthalen-1-yl)thiophen-2-yl)-1H-tetrazole(DNM0577)

A round-bottomed flask was charged with5-(4-chlorobenzyl)-4-(4-methylnaphthalen-1-yl)thiophene-2-carbonitrile(170 mg, 0.45 mmol), zinc bromide (338 mg, 1.50 mmol) and sodium azide(97.5 mg, 1.50 mmol). After degassed, DMF (3 mL) was added. The reactionmixture was heated to 110° C. and stirred at this temperature untilcomplete. The reaction was cooled to rt and 30 mL of 0.1 N aqueous HClwas added. The reaction mixture was extracted with ethyl acetate. Theorganic phase was dried over anhydrous sodium sulphate and thenconcentrated. The residue was purified by flash chromatography(hexane:EtOAc:AcOH=30:10:1). 161 mg (85%) of product was obtained as awhite solid, ¹H NMR (CDCl₃, 500 MHz) δ 8.05 (d, J=8.46 Hz, 1H), 7.73 (s,1H), 7.63 (d, J=8.36 Hz, 1H), 7.53 (t, J=7.58 Hz, 1H), 7.42 (t, J=7.53Hz, 1H), 7.33 (d, J=7.11 Hz, 1H), 7.23 (d, J=7.08 Hz, 1H), 7.14 (d,J=8.25 Hz, 2H), 6.92 (d, J=8.14 Hz, 2H), 3.86 (ABq, 2H, Δδ_(AB)=0.06,J_(AB)=15.90 Hz), 2.70 (s, 3H); ¹³C NMR (CDCl₃, 125 MHz) δ 152.61,145.54, 139.59, 137.96, 135.22, 132.97, 132.76, 132.69, 132.27, 131.09,130.05, 128.81, 127.55, 126.33, 126.25, 126.20, 126.16, 124.75, 122.29,34.24, 19.77.

The following compounds were also prepared using an analogous method:

5-(5-(4-Chlorobenzyl)-4-(4-chlorophenyl)thiophenyl)-1H-tetrazole(DNM0579)

¹H NMR (DMSO, 500 MHz) δ 7.80 (s, 1H), 7.57 (d, J=8.70 Hz, 2H), 7.53 (d,J=8.70 Hz, 2H), 7.40 (d, J=8.34 Hz, 2H), 7.26 (d, J=8.34 Hz, 2H), 4.30(s, 2H); ¹³C NMR (DMSO, 125 MHz) δ 143.21, 138.47, 138.38, 133.64,132.51, 131.51, 130.65, 130.49, 130.24, 128.89, 128.65, 122.97, 32.99.

5-(5-(4-Chlorobenzyl)-4-(4-chloro-2-iso-propylphenyl)thiophenyl)-1H-tetrazole(DNM0580)

¹H NMR (CDCl₃, 500 MHz) δ 7.66 (s, 1H), 7.31 (d, J=2.05 Hz, 1H), 7.19(d, J=8.35 Hz, 2H), 7.16 (dd, J₁=8.12 Hz, J₂=2.10 Hz, 1H), 6.99 (d,J=8.20 Hz, 1H), 7.23 (d, J=78.35 Hz, 2H), 3.89 (ABq, 2H, Δδ_(AB)=0.04,J_(AB)=14.13 Hz), 2.76 (sep, J=6.85 Hz, 1H), 1.10-0.97 (m, 6H); ¹³C NMR(CDCl₃, 125 MHz) δ 152.60, 149.93, 144.93, 139.52, 137.57, 134.85,132.95, 131.97, 131.93, 131.70, 130.02, 129.01, 126.41, 126.15, 122.28,30.02, 30.85, 24.57, 23.42.

5-(5-(4-Chlorobenzyl)-4-(4-fluoronaphthalen-1-yl)thiophen-2-yl)-1H-tetrazole(DNM0587)

¹H NMR (DMSO, 500 MHz) δ 8.17 (d, J=8.25 Hz, 1H), 7.75-7.69 (m, 2H),7.69-7.62 (m, 2H), 7.53-7.42 (m, 2H), 7.30-7.25 (m, 2H), 7.11-7.06 (m,2H), 4.10-3.91 (m, 2H); ¹³C NMR (CDCl₃, 125 MHz) δ 158.86, 156.86,144.75, 138.33, 137.42, 132.85, 132.81, 131.60, 131.28, 130.30, 128.78,128.75, 128.41, 127.93, 127.85, 127.78, 126.99, 125.40, 125.38, 123.10,122.97, 120.43, 120.39, 109.53, 109.37, 33.01, 21.07.

5-(4-(Biphenyl-2-yl)-5-(4-chlorobenzyl)thiophen-2-yl)-1H-tetrazole(DNM0588)

¹H NMR (DMSO, 500 MHz) δ 7.58-7.52 (m, 1H), 7.52-7.46 (m, 3H), 7.43 (d,J=7.60 Hz, 2H), 7.33-7.24 (m, 5H), 7.20-7.13 (m, 2H), 6.89 (d, J=8.33Hz, 2H), 3.73 (s, 2H); ¹³C NMR (DMSO, 125 MHz) δ 143.34, 140.80, 140.48,139.40, 138.13, 133.20, 131.66, 131.37, 130.76, 130.42, 130.30, 129.03,128.69, 128.42, 128.23, 127.71, 127.03, 122.21, 33.01.

Ethyl 3-(2-(4-chlorobenzyl)-5-(1H-tetrazol-5-yl)thiophen-3-yl)benzoate(DNM0595)

¹H NMR (DMSO, 500 MHz) δ 8.09-7.93 (m, 2H), 7.87 (s, 1H), 7.80 (d,J=7.24 Hz, 1H), 7.66 (1, J=7.71 Hz, 1H), 7.40 (d, J=7.86 Hz, 2H), 7.26(d, J=7.86 Hz, 2H), 4.52-4.14 (m, 4H), 1.31 (t, J=7.07 Hz, 3H); ¹³C NMR(DMSO, 125 MHz) δ 165.42, 143.15, 138.63, 138.45, 135.17, 133.07,131.49, 130.65, 130, 48, 130.40, 129.46, 128.79, 128.64, 128.37, 123.12,60.94, 32.96, 14.11.

1-(3-(2-(4-Chlorobenzyl)-5-(1H-tetrazol-5-yl)thiophen-3-yl)phenyl)butan-1-one(DNM0600)

¹H NMR (DMSO, 500 MHz) δ 8.00-7.97 (m, 2H), 7.88 (s, 1H), 7.77 (d,J=7.70 Hz, 1H), 7.65 (t, J=7.95 Hz, 1H), 7.39 (d, J=8.40 Hz, 2H), 7.26(d, J=8.40 Hz, 2H), 4.32 (s, 2H), 2.98 (t, J=7.15 Hz, 2H), 1.63 (sex,J=7.30 Hz, 1H), 0.93 (t, J=7.35 Hz, 3H); ¹³C NMR (DMSO, 125 MHz) δ199.80, 142.91, 138.94, 138.55, 137.20, 135.19, 132.80, 131.50, 130.68,130.42, 129.40, 128.66, 127.75, 127.08, 123.27, 39.85, 32.99, 17.17,13.61.

5-(4-(3-Butylphenyl)-5-(4-chlorobenzyl)thiophen-2-yl)-1H-tetrazole(DNM0606)

¹H NMR (DMSO, 500 MHz) δ 7.81 (s, 1H), 7.42-7.34 (m, 3H), 7.32-7.26 (m,2H), 7.26-7.21 (m, 3H), 4.30 (s, 2H), 2.62 (t, J=7.65 Hz, 2H), 1.56 (qi,J=7.55 Hz, 2H), 1.31 (sex, J=7.54 Hz, 2H), 0.90 (t, J=7.37 Hz, 3H); ¹³CNMR (DMSO, 125 MHz) δ 142.95, 139.99, 138.72, 134.71, 131.41, 130.87,130.39, 128.77, 128.59, 128.58, 128.34, 127.76, 125.68, 122.74, 34.73,33.04, 21.75, 13.79.

Ethyl3-(2-(4-chlorobenzyl)-5-(1H-tetrazol-5-yl)thiophen-3-yl)-5-hydroxybenzoate(DNM0609)

¹H NMR (DMSO, 500 MHz) δ 10.12 (s, 1H), 7.81 (s, 1H), 7.45 (s, 1H),7.43-7.38 (m, 3H), 7.27 (d, J=8.35 Hz, 2H), 7.14 (s, 1H), 4.34-4.25 (m,4H), 1.30 (t, J=7.10 Hz, 3H); ¹³C NMR (DMSO, 125 MHz) δ 165.41, 157.89,150.98, 142.84, 138.79, 138.53, 136.37, 131.64, 131.47, 130.45, 130.38,128.64, 123.18, 119.84, 119.74, 114.99, 60.87, 32.99, 14.11.

5-(5-(4-Chlorobenzyl)-4-(3-(cyclopentylmethyl)phenyl)thiophen-2-yl)-1H-tetrazole(DNM0610)

¹H NMR (DMSO, 500 MHz) δ 7.81 (s, 1H), 7.41-7.43 (m, 3H), 7.30 (d,J=7.70 Hz, 1H), 7.26 (s, 1H), 7.25-7.19 (m, 3H), 4.30 (s, 2H), 2.61 (d,J=7.45 Hz, 2H), 2.10-2.01 (m, 1H), 1.67-1.54 (m, 4H), 1.54-1.41 (m, 2H),1.21-1.09 (m, 2H); ¹³C NMR (DMSO, 125 MHz) δ 150.80, 142.42, 142.22,140.05, 138.70, 134.61, 131.41, 130.92, 130.34, 128.70, 128.67, 128.58,128.11, 125.68, 122.65, 41.32, 41.17, 33.01, 31.90, 24.48.

3-(2-(4-Chlorobenzyl)-5-(1H-tetrazol-5-yl)thiophen-3-yl)phenol (DNM0613)

¹H NMR (DMSO, 500 MHz) δ 9.65 (s, 1H), 7.65 (s, 1H), 7.40 (d, J=8.35 Hz,2H), 7.29 (t, J=7.88 Hz, 1H), 7.26 (d, J=8.40 Hz, 2H), 6.91 (d, J=7.90Hz, 1H), 6.87 (s, 1H), 6.82 (dd, J₁=1.79 Hz, J₂=8.10 Hz, 1H), 4.29 (s,2H); ¹³C NMR (DMSO, 125 MHz) δ 157.66, 142.45, 139.94, 138.72, 136.01,131.46, 130.76, 130.44, 129.94, 128.64, 119.11, 115.20, 114.78, 33.07.

5-(5-(4-Chlorobenzyl)-4-(3-methoxyphenyl)thiophen-2-yl)-1H-tetrazole(DNM0615)

¹H NMR (DMSO, 500 MHz) δ 7.84 (s, 1H), 7.47-7.40 (m, 3H), 7.29 (d,J=8.45 Hz, 2H), 7.09 (d, J=7.80 Hz, 1H), 7.06-7.04 (m, 1H), 7.02 (dd,J₁=2.00 Hz, J₂=8.20 Hz, 1H), 4.34 (s, 2H), 3.81 (s, 3H); ¹³C NMR (DMSO,125 MHz) δ 159.49, 142.69, 139.68, 138.70, 136.13, 131.45, 130.87,130.44, 129.99, 128.65, 120.68, 113.83, 113.50, 55.14, 33.06.

5-(5-(4-Chlorobenzyl)-4-(3-butoxyphenyl)thiophen-2-yl)-1H-tetrazole(DNM0616)

¹H NMR (DMSO, 500 MHz) δ 7.81 (s, 1H), 7.44-7.36 (m, 3H), 7.26 (d,J=8.35 Hz, 2H), 7.05 (d, J=7.60 Hz, 1H), 6.99-6.95 (m, 2H), 4.31 (s,2H), 3.95 (t, J=6.50 Hz, 2H), 1.69 (pen, J=6.96 Hz, 2H), 1.43 (sex,J=7.50 Hz, 2H), 0.94 (t, J=7.40 Hz, 2H); ¹³C NMR (DMSO, 125 MHz) δ158.90, 150.96, 142.40, 139.75, 138.74, 136.10, 131.43, 130.85, 130.39,129.96, 128.63, 122.80, 120.51, 114.13, 67.14, 33.04, 30.73, 18.72,13.72.

5-(5-(4-Chlorobenzyl)-4-(3-ethoxyphenyl)thiophen-2-yl)-1H-tetrazole(DNM0617)

¹H NMR (DMSO, 500 MHz) δ 7.81 (s, 1H), 7.44-7.37 (m, 3H), 7.26 (d,J=8.40 Hz, 2H), 7.05 (d, J=7.65 Hz, 1H), 7.01-6.95 (m, 2H), 4.31 (s,2H), 4.04 (q, J=7.00 Hz, 2H), 1.33 (t, J=7.00 Hz, 3H); ¹³C NMR (DMSO,125 MHz) δ 158.74, 142.67, 139.70, 138.71, 136.11, 131.44, 130.89,130.43, 130.01, 128.63, 120.54, 114.20, 114.01, 63.05, 33.05, 14.63.

5-(5-(4-Chlorobenzyl)-4-(3-propoxyphenyl)thiophen-2-yl)-1H-tetrazole(DNM0618)

¹H NMR (DMSO, 500 MHz) δ 7.82 (s, 1H), 7.43-7.36 (m, 3H), 7.26 (d,J=8.40 Hz, 2H), 7.05 (d, J=7.65 Hz, 1H), 7.00-6.95 (m, 2H), 4.31 (s,2H), 3.91 (t, J=6.60 Hz, 2H), 1.73 (sex, 7.06, 2H), 0.98 (t, J=7.40 Hz,3H); ¹³C NMR (DMSO, 125 MHz) δ 158.88, 142.50, 139.74, 138.73, 136.09,131.43, 130.91, 130.39, 129.97, 128.63, 122.72, 120.51, 114.14, 68.92,33.04, 22.02, 10.37.

Example 5 Synthesis of DNM0461 and Analogues

5-(3,5-Dibromophenyl)-1H-tetrazole

A round-bottomed flask was charged with 3,5-dibromobenzonitrile (15.65g, 60.00 mmol), sodium azide (7.80 g, 120.00 mmol) and zinc bromide(27.00 g, 120.00 mmol). After degassed, DMF (100 mL) was added. Thereaction mixture was heated to 120° C. and stirred at this temperatureuntil complete. The reaction was cooled to rt and then in an ice-waterbath. 300 mL of 1N aqueous HCl was added. The white precipitate formedwas collected by suction filtration, washed with water, and driedtogether with phosphorous pentoxide under vacuum to afford 17.32 g (95%)of product.

2-Benzhydryl-5-(3,5-dibromophenyl)-2H-tetrazole

A suspension of 5-(3,5-dibromophenyl)-1H-tetrazole (6.08 g, 20.00 mmol),diphenylmethanol (3.68 g, 20.00 mmol) and TsOH.H₂O (0.38 g, 2.00 mmol)in toluene (50.0 mL) was heated to 100° C. until a clear solution wasobtained. The solution was cooled to rt, and filtered. The filtrationwas concentrated, and the residue was purified by recrystallization inether and hexane to afford 8.00 g (85%) of product as a white solid.

2-Benzhydryl-5-(3,5-bis(4-methylnaphthalen-1-yl)phenyl)-2H-tetrazole

A round-bottomed flask was charged with2-benzhydryl-5-(3,5-dibromophenyl)-2H-tetrazole (282 mg, 0.60 mmol),4-methylnaphthalen-1-ylboronic acid (251 mg, 1.32 mmol) and Pd(PPh₃)₄(35 mg, 0.03 mmol). After degassed, dioxane (5 mL) and aqueous sodiumcarbonate (3 mL, 2M, 6.0 mmol) was added. The reaction mixture washeated to 90° C. until the reaction was complete. 30 mL of water wasadded, and the reaction mixture was extracted with ethyl acetate. Theorganic phase was dried over anhydrous sodium sulphate and thenconcentrated. The residue was purified by flash chromatography(hexane:CH₂Cl₂=2:1) to afford 0.32 g (90%) of product as a white solid.

5-(3,5-Bis(4-methylnaphthalen-1-yl)phenyl)-2H-tetrazole (DNM0461)

2-Benzhydryl-5-(3,5-bis(4-methylnaphthalen-1-yl)phenyl)-2H-tetrazole(0.32 g, 0.54 mmol) was dissolved in dichloromethane (2 mL). Anisole(0.3 mL, 2.76 mmol) and TFA (1 mL) were added sequentially. The reactionwas stirred overnight, and then concentrated. The residue was purifiedby flash chromatography (hexane:ethyl acetate:AcOH=30:10:1) to afford0.22 g (95.5%) of product as a white solid, ¹H NMR (DMSO, 500 MHz) δ8.24 (d, J=1.55 Hz, 2H), 8.25 (d, J=8.15 Hz, 2H), 8.03 (d, J=8.20 Hz,2H), 7.72 (s, 1H), 7.65 (t, J=7.58 Hz, 2H), 7.60 (t, J=7.44 Hz, 2H),7.56 (d, J=7.15 Hz, 2H), 7.51 (d, J=7.29 Hz, 2H), 2.74 (s, 6H); ¹³C NMR(DMSO, 125 MHz) δ 141.58, 136.38, 134.51, 133.81, 132.43, 130.72,127.15, 127.03, 126.51, 126.32, 126.11, 125.61, 124.76.19.21.

The following compounds were also prepared using an analogous method:

5-(2,5-Bis(4-methylnaphthalen-1-yl)phenyl)-1H-tetrazole (DNM0446)

¹H NMR (DMSO, 500 MHz) δ 8.16 (d, J=8.45 Hz, 1H), 8.06 (d, J=8.41 Hz,2H), 7.97 (s, 1H), 7.80 (dd, J₁=7.84 Hz, J₂=1.80 Hz, 1H), 7.69-7.49 (m,7H), 7.42 (t, J=7.56 Hz, 1H), 7.39 (d, J=7.09 Hz, 1H), 7.28 (d, J=7.09Hz, 1H), 2.73 (s, 3H), 2.70 (s, 3H); ¹³C NMR (DMSO, 125 MHz) δ 139.96,138.93, 136.37, 135.51, 134.38, 133.98, 132.46, 132.21, 132.06, 132.03,131.37, 130.98, 130.80, 127.11, 127.00, 126.43, 126.34, 126.10, 126.04,125.89, 125.76, 125.73, 124.73, 124.45, 19.23, 19.14.

5-(2,5-Bis(4-chloro-2-methylphenyl)phenyl)-1H-tetrazole (DNM0447)

¹H NMR (DMSO, 500 MHz) δ 7.79 (s, 1H), 7.65 (d, J=7.16 Hz, 1H),7.51-7.43 (m, 2H), 7.38 (s, 2H), 7.33 (s, 1H), 7.22 (d, J=8.09 Hz, 1H),7.05 (d, J=8.14 Hz, 1H), 2.33 (s, 3H), 1.97 (s, 3H); ¹³C NMR (DMSO, 125MHz) δ 139.71, 138.72, 132.58, 138.20, 138.13, 137.59, 132.41, 132.10,131.39, 131.20, 131.08, 130.96, 130.10, 130.05, 129.00, 126.06, 125.39,20.05, 19.56.

5-(3,5-Bis(4-chlorophenyl)phenyl)-2H-tetrazole (DNM0470)

¹H NMR (DMSO, 500 MHz) δ 8.34 (d, J=1.69 Hz, 2H), 8.16 (d, J=1.69 Hz,2H), 7.95-7.91 (m, 4H), 7.65-7.61 (m, 4H); ¹³C NMR (DMSO, 125 MHz) δ140.76, 137.76, 133.22, 129.07, 128.94, 127.58, 124.42.

5-(4′-Chloro-5-(4-methylnaphthalen-1-yl)biphenyl-3-yl)-1H-tetrazole(DNM0480)

¹H NMR (DMSO, 500 MHz) δ 8.42 (t, J=1.60 Hz, 1H), 8.16 (d, J=7.98 Hz,1H), 8.14 (t, J=1.58 Hz, 1H), 7.95 (t, J=1.62 Hz, 1H), 7.92 (d, J=7.90Hz, 1H), 7.90 (d, J=8.70 Hz, 2H), 7.67-7.63 (m, 1H), 7.61 (d, J=8.70 Hz,2H), 7.59-7.55 (m, 1H), 7.54-7.50 (m, 2H), 2.76 (s, 3H); ¹³C NMR (CDCl₃,125 MHz) δ 143.14, 141.32, 138.28, 137.09, 134.98, 134.46, 133.04,131.81, 131.53, 129.36, 128.72, 128.11, 127.06, 126.37, 126.31, 126.26,126.08, 125.13, 124.79, 19.84.

5-(3,5-Bis(4-fluoronaphthalen-1-yl)phenyl)-2H-tetrazole (DNM0539)

¹H NMR (DMSO, 500 MHz) δ 8.26 (d, J=1.55 Hz, 2H), 8.19 (dd, J₁=8.15 Hz,J₂=1.87 Hz, 2H), 8.05 (d, J=7.87 Hz, 2H), 7.77 (t, J=1.55 Hz, 1H),7.75-7.65 (m, 6H), 7.49 (dd, J₁=10.60 Hz, J₂=7.95 Hz, 2H); ¹³C NMR(DMSO, 125 MHz) δ 158.81, 156.81, 140.71, 134.46, 134.42, 133.80,132.00, 131.97, 128.67, 127.55, 127.48, 127.46, 126.96, 125.44, 125.42,123.15, 123.02, 120.46, 120.42, 109.60, 109.44.

Example 6 Synthesis of DNM0566 and Analogues

Ethyl 4,5-dibromothiophene-2-carboxylate

To a stirred solution of ethyl thiophene-2-carboxylate (12.62 g, 80.8mmol) in 12 mL of sulfuric acid and 40 mL of TFA was added NBS (32.00 g,177.8 mmol) in portions during 2˜3 hours. After stirred overnight atroom temperature, the reaction mixture was poured onto ice. The whiteprecipitate formed was collected by suction filtration, and purified byrecrystallization in methanol. 23.38 g (92%) of product was obtained asa white solid, mp: 47.0-48.0 (lit. mp 48.0-49.0° C., Bull. Chem. Soc.Jpn. 1991, 64 (8), 2566-8).

Ethyl 4,5-bis(4-chloro-2-isopropylphenyl)thiophene-2-carboxylate

A round-bottomed flask was charged with ethyl4,5-dibromothiophene-2-carboxylate (235.5 mg, 0.75 mmol),4-chloro-2-isopropylphenyboronic acid (327.5 mg, 1.65 mmol) andPd(PPh₃)₄ (43 mg, 0.0375 mmol). After degassed, dioxane (5 mL) andaqueous sodium carbonate (3 mL, 2M, 6.0 mmol) was added. The reactionmixture was heated to 90° C. until the reaction was complete. Aftercooled to room temperature, the reaction mixture was diluted with 30 mLof water and then extracted with ethyl acetate. The organic phase wasdried over anhydrous sodium sulphate and then concentrated. The residuewas purified by flash chromatography (hexane:CH₂Cl₂=2:1) to afford 0.28g (81%) of product as a white solid.

4,5-Bis(4-chloro-2-isopropylphenyl)thiophene-2-carboxylic acid (DNM0566)

To a solution of ethyl4,5-bis(4-chloro-2-isopropylphenyl)thiophene-2-carboxylate (280 mg, 0.61mmol) in THF (3 mL) and MeOH (3 mL) was added a solution of LiOH (72 mg,3.0 mmol) in water (2 mL). The reaction was stirred at rt untilcomplete, and then concentrated. The residue was re-dissolved in 20 mLof water and acidified with 1N aq. HCl to pH 2. The white precipitatewas extracted with ethyl acetate. The organic phase was dried overanhydrous sodium sulphate and then concentrated. The residue waspurified by recrystallization in a mixture solvent of ethyl acetate andhexane to afford 0.24 g (91%) of product as a white solid, ¹H NMR(CDCl₃, 500 MHz) δ 7.78 (s, 1H), 7.23 (d, J=2.15 Hz, 1H), 7.22 (d,J=1.88 Hz, 1H), 7.13-7.07 (m, 2H), 7.03 (dd, J₁=8.23 Hz, J₂=2.20 Hz,1H), 6.89 (d, J=8.26 Hz, 1H), 2.95 (m, 2H), 1.04 (d, J—4.94 Hz, 6H),0.98 (d, J=6.95 Hz, 6H); ¹³C NMR (CDCl₃, 125 MHz) δ 167.13, 149.87,149.06, 146.58, 139.45, 137.21, 135.53, 134.39, 132.79, 131.84, 131.57,131.14, 129.01, 126.43, 126.28, 125.86, 125.77, 30.27, 30.23, 23.93.

The following compounds were also prepared using an analogous method:

4,5-Bis(4-biphenyl)thiophene-2-carboxylic acid (DNM0497)

¹H NMR (CDCl₃, 500 MHz) δ 13.34 (s, 1H), 7.86 (s, 1H), 7.72-7.65 (m,8H), 7.50-7.32 (m, 10H); ¹³C NMR (CDCl₃, 125 MHz) δ 162.69, 143.94,140.16, 139.30, 139.09, 139.00, 138.17, 135.56, 134.08, 133.00, 131.98,129.46, 129.38, 129.02, 128.98, 127.87, 127.63, 127.12, 126.86, 126.61,126.53.

4,5-Bis(4-chlorophenyl)thiophene-2-carboxylic acid (DNM0498)

¹H NMR (CDCl₃, 500 MHz) δ 13.39 (s, 1H), 7.81 (s, 1H), 7.46 (d, J=8.40Hz, 2H), 7.42 (d, J=8.40 Hz, 2H), 7.32 (d, J=8.50 Hz, 2H), 7.29 (d,J=8.42 Hz, 2H); ¹³C NMR (CDCl₃, 125 MHz) δ 162.53, 143.06, 137.68,135.30, 133.60, 133.47, 133.44, 132.47, 131.46, 130.74, 130.64, 129.13,128.78.

4,5-Bis(5-chloro-2-methylphenyl)thiophene-2-carboxylic acid (DNM0501)

¹H NMR (CDCl₃, 500 MHz) δ 13.38 (s, 1H), 7.77 (s, 1H), 7.36 (d, J=2.20Hz, 1H), 7.34 (dd, J₁=2.20 Hz, J₂=8.15 Hz, 1H), 7.26-7.22 (m, 3H), 7.05(s, 1H), 2.01 (s, 3H), 1.96 (s, 3H); ¹³C NMR (CDCl₃, 125 MHz) δ 162.63,143.31, 138.84, 136.36, 135.58, 134.85, 134.75, 133.77, 133.76, 132.18,132.08, 130.47, 130.07, 129.86, 129.60, 128.88, 127.74, 19.22, 19.09.

4,5-Bis(3-chlorophenyl)thiophene-2-carboxylic acid (DNM0502)

¹H NMR (CDCl₃, 500 MHz) δ 13.34 (s, 1H), 7.85 (s, 1H), 7.47 (d, J=8.10Hz, 1H), 7.44-7.34 (m, 5H), 7.26 (d, J=7.65 Hz, 1H), 7.20 (d, J=7.45 Hz,1H); ¹³C NMR (CDCl₃, 125 MHz) δ 162.50, 142.92, 137.70, 136.57, 135.23,134.51, 133.77, 133.50, 133.31, 130.87, 130.48, 128.75, 128.56, 128.53,127.85, 127.69, 127.63.

4,5-Bis(2,4-dimethylphenyl)thiophene-2-carboxylic acid (DNM0503)

¹H NMR (CDCl₃, 500 MHz) δ 13.19 (s, 1H), 7.66 (s, 1H), 7.12 (d, J=8.25Hz, 1H), 7.03-6.95 (m, 3H), 6.87-6.81 (m, 2H), 2.24 (s, 3H), 2.22 (s,3H), 2.07 (s, 3H), 1.93 (s, 3H); ¹³C NMR (CDCl₃, 125 MHz) δ 162.82,144.83, 139.77, 138.16, 136.78, 136.10, 135.28, 135.22, 132.49, 132.04,130.98, 130.91, 130.02, 129.43, 126.45, 126.27, 20.66, 20.61, 19.78,19.64.

4,5-Bis(4-chloro2-methylphenyl)thiophene-2-carboxylic acid (DNM0561)

¹H NMR (CDCl₃, 500 MHz) δ 7.81 (s, 1H), 7.17 (d, J=2.02 Hz, 1H), 7.14(s, 1H), 7.13-7.11 (m, 2H), 7.03 (dd, J₁=8.24 Hz, J₂=2.02 Hz, 1H), 6.87(d, J=8.24 Hz, 1H), 2.12 (s, 3H), 2.03 (s, 3H); ¹³C NMR (CDCl₃, 125 MHz)δ 167.74, 147.16, 140.15, 139.20, 138.26, 137.56, 135.29, 134.06,133.61, 132.85, 131.97, 131.74, 131.13, 131.03, 130.88, 126.50, 126.43,20.61, 20.59.

Example 7 Synthesis of DNM0631 and Analogues

2-Bromo-3-methylthiophene

NBS (8.90 g, 50.0 mmol) was added portion wise to a stirred solution of3-methylthiophene (4.90 g, 50.0 mmol) in acetic acid (20 mL) at rt.After complete addition, the reaction was stirred at rt until it wascomplete. The reaction mixture was poured into ice-water, and thenextracted with a 3:1 mixture solvent of hexane and ether. The organiclayer was washed with 1N aq. NaOH and brine. After dried over anhydroussodium sulphate, the organic phase was concentrated on vacuum to afford2-bromo-3-methylthiphene (8.20 g, 92.7%), which was used directly in thenext step without further purification.

3-Methylthiophene-2-carbonitrile

A round-bottomed flask was charged with 2-bronco-3-methylthiphene (8.20g, 46.3 mmol) and CuCN (8.29 g, 92.6 mmol). After degassed, 50 mL of DMFwas added. The reaction mixture was heated to 150° C. overnight. Aftercooled to room temperature, the reaction was quenched with 250 mL ofwater, and extracted with ethyl acetate. The organic phase was driedover anhydrous sodium sulphate and then concentrated. The residue waspassed through a short and fat silicon column, and the column was elutedwith hexane to afford 5.07 g of product.

4,5-Dibromo-3-methylthiophene-2-carbonitrile

Bromine (4.25 mL, 82.6 mmol) was added drop wise to a stirred solutionof 3-methylthiophene-2-carbonitrile (5.07 g, 41.2 mmol) in DMF (20 mL)at rt. After complete addition, the reaction was heated to 60° C. untilit was complete. After cooled to rt, the reaction mixture was pouredinto ice-water. The light yellow solid formed was collected throughsuction filtration, and dried together with P₂O₅ under vacuum to afford9.84 g of 4,5-dibromo-3-methylthiophene-2-carbonitrile.

4,5-Bis(4-chloro-2-isopropylphenyl)-3-methylthiophene-2-carbonitrile

A round-bottomed flask was charged with4,5-dibromo-3-methylthiophene-2-carbonitrile (140.5 mg, 0.50 mmol),2-(4-chloro-2-isopropylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(308.7 mg, 1.10 mmol) and Pd(PPh₃)₄ (29 mg, 0.025 mmol). After degassed,dioxane (5 mL) and aqueous sodium carbonate (3 mL, 2M, 6.0 mmol) wasadded. The reaction mixture was heated to 95° C. until the reaction wascomplete. After cooled to room temperature, the reaction mixture wasdiluted with 30 mL of water and then extracted with ethyl acetate. Theorganic phase was dried over anhydrous sodium sulphate and thenconcentrated. The residue was purified by flash chromatography(hexane:CH₂Cl₂=4:1) to afford 190.5 mg (89%) of product as a whitesolid.

5-(4,5-Bis(4-chloro-2-isopropylphenyl)-3-methylthiophen-2-yl)-1H-tetrazole(DNM0631)

A round-bottomed flask was charged with4,5-bis(4-chloro-2-isopropylphenyl)-3-methylthiophene-2-carbonitrile(190.5 mg, 0.44 mmol), zinc bromide (338 mg, 1.50 mmol) and sodium azide(97.5 mg, 1.50 mmol). After degassed, DMF (3 mL) was added. The reactionmixture was heated to 130° C. and stirred at this temperature untilcomplete. The reaction was cooled to rt and 30 mL of 0.1 N aqueous HClwas added. The reaction mixture was extracted with ethyl acetate. Theorganic phase was dried over anhydrous sodium sulphate and thenconcentrated. The residue was purified by flash chromatography(hexane:EtOAc:AcOH=30:10:1). 188.7 mg (90%) of product was obtained as awhite solid, ¹H NMR (CDCl₃, 500 MHz) δ 7.25-7.22 (m, 2H), 7.18 (dd,J₁=8.18 Hz, J₂=2.13 Hz, 1H), 7.09-7.06 (m, 2H), 7.02 (d, J=8.20 Hz, 1H),3.04 (sep, J=6.75 Hz, 1H), 2.74 (sep, J=6.80 Hz, 1H), 2.37 (s, 3H), 1.12(d, J=6.80 Hz, 3H), 1.05 (d, J=6.80 Hz, 6H), 0.90 (d, J=6.80 Hz, 3H);¹³C NMR (CDCl₃, 125 MHz) δ 150.32, 149.87, 141.11, 140.81, 140.72,135.33, 134.47, 132.72, 132.02, 131.51, 129.01, 126.30, 126.20, 125.84,125.52, 30.35, 30.10, 24.76, 23.60, 23.13, 15.78.

The following compounds were also prepared using an analogous method:

5-(4,5-bis(4-chloro-2-methylphenyl)-3-methylthiophen-2-yl)-1H-tetrazole(DNM0614)

¹H NMR (CDCl₃, 500 MHz) δ 7.15 (s, 1H), 7.11 (s, 1H), 7.08 (d, J=8.20Hz, 1H), 7.05-7.01 (m, 2H), 6.93 (d, J=8.30 Hz, 1H), 2.31 (s, 3H), 2.17(s, 3H), 2.04 (s, 31-1); ¹³C NMR (CDCl₃, 125 MHz) δ 141.47, 140.34,139.95, 139.41, 139.16, 134.57, 133.83, 133.81, 132.76, 132.20, 131.43,130.52, 130.42, 126.20, 126.00, 20.81, 20.03, 15.56.

5-(4,5-Bis(4-methylnaphthalen-1-yl)-3-methylthiophen-2-yl)-1H-tetrazole(DNM0620)

¹H NMR (CDCl₃, 500 MHz) δ 8.04 (d, J=8.30 Hz, 1H), 7.91 (d, J=8.30 Hz,1H), 7.86 (d, J=8.40 Hz, 1H), 7.77 (dd, J₁=8.30 Hz, J₂=1.10 Hz, 1H),7.48-7.37 (m, 3H), 7.32 (t, J=7.65 Hz, 1H), 7.18 (d, J=7.20 Hz, 1H),7.06-7.00 (m, 3H), 2.55 (s, 3H), 2.54 (s, 3H), 2.28 (s, 3H); ¹³C NMR(CDCl₃, 125 MHz) δ 142.29, 141.77, 141.39, 135.21, 134.23, 132.73,132.52, 132.44, 132.35, 131.52, 128.93, 128.72, 127.94, 126.61, 126.30,126.07, 125.82, 125.74, 125.70, 125.64, 125.52, 124.44, 124.10, 119.63,19.49, 19.42, 15.54.

5-(4,5-Bis(3-butylphenyl)-3-methylthiophen-2-yl)-1H-tetrazole (DNM0627)

¹H NMR (CDCl₃, 500 MHz) δ 7.30 (t, J=7.53 Hz, 1H), 7.17 (d, J=7.70 Hz,1H), 7.15 (t, J=7.60 Hz, 1H), 7.10-6.97 (m, 5H), 2.59 (t, J=7.58 Hz,2H), 2.48 (s, 3H), 2.46 (t, J=7.75 Hz, 2H), 1.57-1.49 (m, 2H), 1.45-1.37(m, 2H), 1.33-1.18 (m, 4H), 0.90 (t, J=7.35 Hz, 3H), 0.89 (t, J=7.30 Hz,3H); ¹³C NMR (CDCl₃, 125 MHz) δ 143.22, 143.19, 142.95, 141.28, 141.17,135.74, 133.10, 130.46, 129.16, 128.47, 128.30, 128.15, 127.67, 127.45,126.23, 125.52, 35.42, 35.36, 33.56, 33.26, 30.31, 22.15, 22.07, 15.64,13.92.

5-(4,5-Bis(4-fluoronaphthalen-1-yl)-3-methylthiophen-2-yl)-1H-tetrazole(DNM0628)

¹H NMR (CDCl₃, 500 MHz) δ 8.10-8.06 (m, 1H), 8.03 (d, J=8.30 Hz, 1H),7.96 (d, J=8.50 Hz, 1H), 7.76-7.71 (m, 1H), 7.56-7.44 (m, 3H), 7.39 (t,J=7.50 Hz, 1H), 7.22 (dd, J₁=5.40 Hz, J₂=7.80 Hz, 1H), 7.08 (dd, J₁=5.38Hz, J₂=7.83 Hz, 1H), 6.95-6.87 (m, 2H), 2.34 (s, 3H); ¹³C NMR (CDCl₃,125 MHz) δ 159.96, 159.45, 157.93, 157.44, 141.79, 141.44, 133.85,133.81, 133.52, 133.49, 128.95, 128.89, 128.71, 128.67, 128.08, 128.02,127.36, 127.24, 126.30, 126.20, 125.67, 125.53, 125.39, 123.72, 123.59,123.56, 123.43, 121.05, 121.01, 120.70, 120.66, 109.03, 108.87, 108.77,108.61, 15.53.

Example 8 Measurement of AcpS Inhibition

Materials

[³H]Acetyl-CoA was mixed as 1 volume of Perkin Elmer NET290 radiolabeledstock (e.g. 0.1 mCi/ml, 3.7 Ci/mmol in Na-acetate, pH 4.5-5.0=27 μM)with 1.2 volumes of 1 mM unlabelled acetyl-CoA (Sigma). Trichloroaceticacid was prepared as a 10% w/v solution. Bovine serum albumin wasprepared as a 25 mg/ml solution in water. DTT was prepared as a 50 mMsolution in water.

Procedure

-   1. A standard reaction contained the following in a total volume of    10 μl:

Stock reagents Volume (μl) Final concentration 1M Na-phosphate, pH 7 0.550 mM 0.1M MgCl₂ 1 10 mM 50 mM DTT 1  5 mM apo-acyl carrier protein(ACP)    4.4 μg 50 μM AcpS enzyme ~0.06 μg 570 μM [³H]Acetyl-CoA 1 57 μMTest compound e.g. DNM0488 0.5 predetermined μM e.g. 500 or 50 ddH₂O to10 μl

-   2. Tubes were incubated at RT. [³H]acetyl-CoA was added last to    start the reaction. To stop the reaction, 2 μl was removed to a    1.5-ml microfuge tube containing 800 μl cold 10% TCA. Up to four    time points were collected for each reaction, e.g. at 5 min, 10 min,    30 min, and 60 min.-   3. 20 μl of 25 mg/ml BSA was added to each tube, mixed and incubated    on ice for 10 min, then centrifuged at 12,000 g for 5 min to form a    pellet. Supernatant was removed using P1000.-   4. Each pellet was washed twice with 800 μl of cold 10% TCA each    time, then centrifuged at 12,000 g for 5 min after each wash. The    supernatant was discarded. Each pellet was resuspended in 50 μl of    formic acid. The suspension was transferred to a scintillation vial    and radioactivity was measured in 2 ml of liquid scintillation    cocktail.

The random (stochastic) error in scintillation counting is proportionalto the square root of the counts (not dpm) accumulated in the countingperiod; i.e. 10% for 100, 3.3% for 1,000, 1% for 10,000, etc. Providedradioactive counts (dpm) are in the thousands, the assay provides usefuldata. Results are reported in terms of % activity with respect to theblank-subtracted counts for working substrate. A % activity of less thanabout 40% is indicative of AcpS enzyme inhibition by the test compoundat that concentration.

For results, see the table in the Example below.

Example 9 Measurement of Minimum Inhibitory Concentrations (MICs)

Susceptibilities to the compounds were determined using the NationalCommittee for Clinical Laboratory Standards (NCCLS) M7-A6 brothmicrodilution method. Cation-adjusted Mueller-Hinton broth (Ca²⁺, 25μg/mL; Mg²⁺, 12.5 μg/mL) microdilution panels were prepared to containantimicrobial doubling dilution concentrations of an appropriate range.DMSO (dimethylsulfoxide) controls were incorporated into the panel tomimic the quantity of DMSO used in dissolving some of the compounds atthe higher concentrations. Each final panel well volume was 100 μL witha bacterial inoculum of 5×10⁵ CFU (colony forming units)/mL of therelevant bacterium. Panels were read following 16 to 20 h of incubationat 35 degrees Celsius in ambient air. The MIC (minimum inhibitoryconcentration) was defined as the lowest concentration of antimicrobialinhibiting visible growth.

The following table, as well as FIG. 1, indicates results of experimentsof both Examples 8 and 9 where available, demonstrating AcpS inhibitionand antimicrobial effects on Methicillin-Resistant Staph. aureus (MRSA)and E. coli D22:

% AcpS activity % AcpS MIC MIC E Compound @ 500 activity MRSA coli D22ID uM at 50 uM (uM) (uM) DNM0486 0.16 0.82 2 >125 DNM0487 0.08 3.348 >125 DNM0488 0.32 11.49 4 125 DNM0489 0 17.89 2 >125 DNM0504 0.16 7.33125 125 DNM0508 0.1 44.92 4 >125 DNM0509 0.2 31.07 15 >125 DNM0512 0.737.29 8 >125 DNM0531 4.38 96.76 60 >125 DNM0534 0 33.98 8 >125 DNM05353.48 92.62 125 >125 DNM0536 0.2 8.25 8 125 DNM0537 0.14 1.28 2 125DNM0538 0.58 82.18 30 >125 DNM0541 0.3 63.12 30 >125 DNM0542 0.7 9.582 >125 DNM0546 5.16 37.21 4 >125 DNM0548 0.1 3.1 4 60 DNM0549 0.48 71.3130 >125 DNM0550 0.39 47.83 30 >125 DNM0559 0.5 7.01 2 >125 DNM0560 0.0663.58 15 >125 DNM0563 0.56 15.12 8 125 DNM0564 0.75 47.61 15 >125DNM0565 0.11 22.91 15 >125 DNM0570 0.19 33.7 15 125 DNM0571 0.11 8.198 >125 DNM0575 0.23 5.86 8 >125 DNM0572 0.51 4.41 8 >125 DNM0576 0.155.88 4 >125 DNM0577 0.4 5.54 8 >125 DNM0579 3.02 15.74 8 125 DNM05800.85 1.62 8 125 DNM0587 0 2.13 8 DNM0588 0 2.67 8 DNM0446 25.36 4 >125DNM0447 0.47 32.56 15 >125 DNM0453 23.76 8 >125 DNM0457 0 1.56 8 60DNM0461 0.85 13.39 1 >125 DNM0464 0.28 9.03 1 >125 DNM0470 27.69 4 125DNM0474 0.3 15.08 2 >125 DNM0479 0.1 12.79 2 >125 DNM0480 2.04 7.35 4125 DNM0493 2.7 25.28 4 >125 DNM0510 0.12 8.4 4 >125 DNM0511 0 14.368 >125 DNM0514 0.14 5.73 4 >125 DNM0515 3.44 30.26 2 >125 DNM0516 532.01 2 >125 DNM0520 20.86 2 >125 DNM0529 0.16 91.56 30 >125 DNM05320.22 30.66 60 >125 DNM0539 39.39 8 >125 DNM0648 60.1

Example 10 Measurement of Combination MICs for Evaluation of AntibioticAdjuvant Potential in Ps. aeruginosa

Generally following the procedure of Example 9, MICs of ampicillin,erythromycin, and azithromycin were determined against Ps. aeruginosa.Parallel to this effort, solutions were prepared which were identical.To each of these were added 0.5 μL of stock solution of a given compoundof the present invention, then diluted to produce a final concentrationof 125 μM, 60 μM, and/or 30 μM for determination of adjuvant activity.Compounds were determined to be antibiotic adjuvants if the mixtureproduced a lower MIC than that of the antibiotic agent alone. Resultswere as follows, demonstrating antimicrobial adjuvant effects:

-   -   DNM0487 at 125 μM (standalone MIC>2 mM) produced an up to        60-fold lower MIC for erythromycin.    -   DNM0487 at 125 μM produced an up to 60-fold lower MIC for        ampicillin.    -   DNM0488 at 125 μM produced an up to 60-fold lower MIC for        ampicillin.    -   DNM0501 at 60 μM produced an up to 60-fold lower MIC for        erythromycin.    -   DNM0501 at 60 μM produced an up to 250-fold lower MIC for        ampicillin.    -   DNM0537 at 60 μM produced an up to 60-fold lower MIC for        erythromycin.    -   DNM0537 at 30 μM produced an up to ˜7-fold lower MIC for        erythromycin.    -   DNM0537 at 30 μM produced an up to 250-fold lower MIC for        ampicillin.    -   DNM0548 at 125 μM produced an up to 125-fold lower MIC for        erythromycin.    -   DNM0548 at 125 μM produced an up to 125-fold lower MIC for        ampicillin.

Example 11 Measurement of MICs for Selected Compounds Against a Panel ofChallenge Pathogens

The inocula for each bacterial strain were prepared by picking 5-10distinct colonies from the culture plates and suspending them in theappropriate broth as per Clinical and Laboratory Standards Institute(CLSI) guidelines M07-A8, M24-A or M11-A7 as appropriate. The inoculumwas resuspended by vigorous shaking on a vortex mixer for 15 s. Theturbidity was then adjusted to McFarland standard 0.5 (1-5×10⁶ CFU/ml).The inoculum was further diluted in the appropriate media for MIC teststo give a final inoculum in each well of 2-8×10⁵ CFU/ml. MICs weretested in the appropriate broth in accordance with the appropriate CLSIguidelines.

A stock solution of each compound was prepared at a concentration of1.28 g/L in DMSO. The stock was further diluted in the appropriate mediato give a top starting concentration of 128 mg/L in the assay. 100 μL ofthe appropriate media, as per CLSI guidelines, was dispensed into eachwell in columns 2-12. 200 μL of the each compound solution (at 256 mg/L)was dispensed into each well in column 1. 100 μL aliquots were pipettedfrom column 1 wells and dispensed into column 2 with a multichannelpipette (±2% coefficient of variation) thus diluting two-fold. 100 μLsamples were then pipetted from column 2 wells and dispensed into column3. The process was repeated through to column 10. The final 100 μL ofdiluted drug from column 10 was then discarded. Row 11 acted as apositive control (no compound, but organisms added), Row 12 acted as anegative control (no compound, and no organisms added).

100 μL of the appropriate inoculum suspension in the appropriate media,as per CLSI guidelines, was added to the appropriate wells. Thisresulted in a well containing 200 μL final volume (made up of 100 μLdiluted compound or diluents and 100 μL of inoculum or broth alone). Allplates were incubated in the dark under aerobic or anaerobic conditionsat 30-37° C. for 24-48 hours, according to the appropriate CLSIguideline. Plates were read visually 24-48 hours post inoculation.Endpoints of 100% inhibition were determined (CLSI interpretationendpoints following visual examination).

MIC₅₀, MIC₈₀, and 100% inhibition results are reported below in μg/mL inthe following Tables, indicating each compound tested against a givenspecies and strain:

Enterococcus faecalis Bacillus cereus ATCC29212 NCTC6349 Compound 100%80% 50% 100% 80% 50% DNM0610 1 1 1 0.5 0.5 0.5 DNM0488 0.5 0.5 0.5 ≦0.25≦0.25 ≦0.25 DNM0487 2 2 2 0.5 0.5 0.5 DNM0477 8 4 4 1 1 0.5 DNM0566 0.50.5 0.5 ≦0.25 ≦0.25 ≦0.25 DNM0576 1 1 1 0.5 ≦0.25 ≦0.25 DNM0466 4 4 20.5 ≦0.25 ≦0.25 DNM0465 2 2 2 ≦0.25 ≦0.25 ≦0.25 DNM0508 2 2 2 0.5 0.50.5 DNM0547 1 1 1 ≦0.25 ≦0.25 ≦0.25 DNM0474 1 1 1 ≦0.25 ≦0.25 ≦0.25DNM0606 1 1 1 0.5 0.5 0.5 DNM0537 1 1 0.5 ≦0.25 ≦0.25 ≦0.25 DNM0548 0.250.25 0.25 ≦0.25 ≦0.25 ≦0.25 Vancomycin 4 4 4 2 2 1

Staphylococcus epidermidis Staphylococcus epidermidis NRS122 NRS7Compound 100% 80% 50% 100% 80% 50% DNM0610 16 8 4 2 1 1 DNM0488 2 2 10.5 0.25 0.25 DNM0487 4 4 4 1 1 0.5 DNM0477 16 16 8 1 0.5 0.25 DNM0566 22 1 1 1 1 DNM0576 2 2 2 0.5 0.5 0.25 DNM0466 16 8 4 0.5 0.25 0.25DNM0465 8 8 4 1 0.25 0.25 DNM0508 8 8 4 1 0.5 0.5 DNM0547 16 8 4 1 1 1DNM0474 16 8 4 1 1 0.5 DNM0606 4 4 2 1 0.5 0.5 DNM0537 4 4 4 1 1 0.5DNM0548 1 1 1 0.5 0.5 0.5 Vancomycin 2 1 1 4 4 2

Staphylococcus aureus Staphylococcus aureus NRS2 (ATCC700698) ATCC43300Compound 100% 80% 50% 100% 80% 50% DNM0610 0.5 0.5 0.5 0.5 0.5 0.5DNM0488 0.5 0.5 0.5 0.5 0.5 0.5 DNM0487 2 2 1 1 1 1 DNM0477 2 2 2 2 2 1DNM0566 0.5 0.5 0.25 ≦0.25 ≦0.25 ≦0.25 DNM0576 0.5 0.5 0.5 0.5 0.5 0.5DNM0466 1 1 1 1 1 1 DNM0465 1 1 1 1 1 1 DNM0508 1 1 0.5 1 1 1 DNM0547 11 0.5 0.5 0.5 0.5 DNM0474 0.5 0.5 0.5 0.5 0.5 0.5 DNM0606 0.5 0.5 0.5≦0.25 ≦0.25 ≦0.25 DNM0537 1 1 1 1 1 0.5 DNM0548 0.5 0.5 0.5 ≦0.25 ≦0.25≦0.25 Vancomycin 1 1 0.5 0.5 0.5 0.5

Staphylococcus aureus Staphylococcus aureus NRS1 (ATCC700699) NRS382Compound 100% 80% 50% 100% 80% 50% DNM0610 ≦0.25 ≦0.25 ≦0.25 0.5 0.5 0.5DNM0488 ≦0.25 ≦0.25 ≦0.25 0.5 0.5 0.5 DNM0487 0.5 0.5 0.25 1 1 1 DNM04771 1 1 2 2 2 DNM0566 ≦0.25 ≦0.25 ≦0.25 0.5 0.5 0.5 DNM0576 0.5 0.5 0.250.5 0.5 0.5 DNM0466 1 1 0.5 2 2 1 DNM0465 1 1 1 2 2 2 DNM0508 1 1 0.5 11 1 DNM0547 0.5 0.5 0.5 0.5 0.5 0.5 DNM0474 0.5 0.5 0.25 0.5 0.5 0.5DNM0606 0.5 0.5 0.5 0.5 0.5 0.5 DNM0537 ≦0.25 ≦0.25 ≦0.25 ≦0.25 ≦0.25≦0.25 DNM0548 ≦0.25 ≦0.25 ≦0.25 0.5 0.5 0.5 Vancomycin 4 4 2 1 1 1

Staphylococcus aureus Staphylococcus aureus NRS383 NRS384 Compound 100%80% 50% 100% 80% 50% DNM0610 0.5 0.5 0.5 ≦0.25 ≦0.25 ≦0.25 DNM0488 0.50.5 0.5 0.5 0.5 0.5 DNM0487 1 1 1 1 1 1 DNM0477 4 4 2 2 2 2 DNM0566≦0.25 ≦0.25 ≦0.25 0.5 0.5 0.5 DNM0576 0.5 0.5 0.5 0.5 0.5 0.5 DNM0466 22 1 1 1 1 DNM0465 2 2 1 2 2 1 DNM0508 1 1 1 1 1 1 DNM0547 0.5 0.5 0.5 11 1 DNM0474 0.5 0.5 0.5 0.5 0.5 0.5 DNM0606 ≦0.25 ≦0.25 ≦0.25 ≦0.25≦0.25 ≦0.25 DNM0537 ≦0.25 ≦0.25 ≦0.25 ≦0.25 ≦0.25 ≦0.25 DNM0548 ≦0.25≦0.25 ≦0.25 ≦0.25 ≦0.25 ≦0.25 Vancomycin 0.5 0.5 0.5 1 1 1

Staphylococcus aureus Staphylococcus aureus EUP SAU040 EUP MRSA004Compound 100% 80% 50% 100% 80% 50% DNM0610 0.5 0.5 0.5 ≦0.25 ≦0.25 ≦0.25DNM0488 0.5 0.5 0.5 0.5 0.5 0.5 DNM0487 2 1 1 2 2 1 DNM0477 2 2 1 2 2 2DNM0566 0.5 0.5 0.5 ≦0.25 ≦0.25 ≦0.25 DNM0576 ≦0.25 ≦0.25 ≦0.25 ≦0.25≦0.25 ≦0.25 DNM0466 2 1 1 2 2 1 DNM0465 1 1 1 1 1 1 DNM0508 1 1 1 1 1 1DNM0547 0.5 0.25 0.25 0.5 0.5 0.5 DNM0474 0.25 0.5 0.5 0.5 0.5 0.5DNM0606 ≦0.25 ≦0.25 ≦0.25 ≦0.25 ≦0.25 ≦0.25 DNM0537 1 1 1 1 1 0.5DNM0548 0.5 0.5 0.5 0.5 0.5 0.5 Vancomycin 1 1 1 0.5 0.5 0.5

Staphylococcus aureus Staphylococcus aureus EUP MRSA002 EUP MRSA002Compound 100% 80% 50% 100% 80% 50% DNM0610 0.5 0.5 0.5 0.5 0.5 0.5DNM0488 1 1 0.5 0.5 0.5 0.5 DNM0487 2 2 1 1 1 1 DNM0477 4 2 2 2 2 1DNM0566 ≦0.25 ≦0.25 ≦0.25 ≦0.25 ≦0.25 ≦0.25 DNM0576 ≦0.25 ≦0.25 ≦0.250.5 0.5 0.5 DNM0466 1 1 1 2 1 1 DNM0465 1 1 1 1 1 0.5 DNM0508 1 1 0.5 11 1 DNM0547 1 1 1 0.5 0.5 0.5 DNM0474 1 1 1 0.5 0.5 0.5 DNM0606 0.5 0.50.5 ≦0.25 ≦0.25 ≦0.25 DNM0537 1 1 1 1 1 0.5 DNM0548 0.5 0.5 0.5 ≦0.25≦0.25 ≦0.25 Vancomycin 0.5 0.5 0.5 0.5 0.5 0.5

Staphylococcus aureus Staphylococcus aureus AUR131 007035 AUR60 7005Compound 100% 80% 50% 100% 80% 50% DNM0610 0.5 0.5 0.5 0.5 0.5 0.5DNM0488 0.5 0.5 0.5 0.5 0.5 0.5 DNM0487 1 1 1 1 1 1 DNM0477 2 2 1 2 2 1DNM0566 ≦0.25 ≦0.25 ≦0.25 0.5 0.5 0.5 DNM0576 0.5 0.5 0.5 0.5 0.5 0.5DNM0466 1 1 1 1 1 1 DNM0465 2 2 1 1 1 1 DNM0508 1 1 1 1 1 1 DNM0547 1 11 1 1 1 DNM0474 0.5 0.5 0.5 0.5 0.5 0.5 DNM0606 0.5 0.5 0.5 0.5 0.5 0.5DNM0537 ≦0.25 ≦0.25 ≦0.25 ≦0.25 ≦0.25 ≦0.25 DNM0548 0.5 0.5 0.5 0.5 0.50.5 Vancomycin 0.5 0.5 0.5 1 1 0.5

Staphylococcus aureus Staphylococcus aureus AUR59 7005 AUR58 7005Compound 100% 80% 50% 100% 80% 50% DNM0610 0.5 0.5 0.5 2 2 1 DNM0488 0.50.5 0.5 1 0.5 0.5 DNM0487 1 1 1 1 1 1 DNM0477 2 2 2 2 2 1 DNM0566 0.50.25 0.25 1 1 1 DNM0576 0.5 0.5 0.5 0.5 0.5 0.5 DNM0466 1 1 1 2 1 1DNM0465 1 1 1 1 1 1 DNM0508 1 1 1 1 1 1 DNM0547 1 1 1 2 2 1 DNM0474 0.50.5 0.5 1 1 0.5 DNM0606 0.5 0.5 0.5 1 1 0.5 DNM0537 ≦0.25 ≦0.25 ≦0.25≦0.25 ≦0.25 ≦0.25 DNM0548 0.5 0.5 0.5 1 1 1 Vancomycin 1 0.5 0.5 1 1 1

Enterococcus faecium Enterococcus faecalis ATCC700221 ATCC51299 Compound100% 80% 50% 100% 80% 50% DNM0610 0.5 0.5 0.5 1 1 1 DNM0488 0.5 0.250.25 0.5 0.5 0.5 DNM0487 1 1 1 2 2 1 DNM0477 4 4 2 4 4 2 DNM0566 0.50.25 0.25 0.5 0.5 0.5 DNM0576 0.5 0.25 0.25 1 1 1 DNM0466 2 2 2 2 2 2DNM0465 2 1 1 2 2 2 DNM0508 1 1 1 2 2 2 DNM0547 1 1 1 0.5 0.5 0.5DNM0474 1 1 1 1 1 1 DNM0606 1 1 1 1 1 1 DNM0537 0.5 0.5 0.5 0.5 0.5 0.5DNM0548 ≦0.25 ≦0.25 ≦0.25 ≦0.25 ≦0.25 ≦0.25Vancomycin >16 >16 >16 >16 >16 >16

These results indicate that the compounds of present invention arepotently antibacterial against a variety of antibiotic-resistantstrains, and in many cases (particularly with respect to VRE), morepotent than vancomycin.

Example 12 Measurement of Minimum Inhibitory Concentrations AgainstStreptococcus pneumoniae Group A

Generally following the procedure of Example 9, but using Todd Hewittbroth (beef heart digest) rather than Mueller-Hinton broth, MICs wereinitially measured against Streptococcus pneumoniae Group A forcompounds of the present invention as follows:

DNM0548 <0.25 μM DNM0576    0.5 μM DNM0620 <0.25 μM DNM0629 <0.25 μMDNM0631 <0.25 μM DNM0636    0.8 μM DNM0640 <0.25 μM

Repeated measurement of MIC of the above seven compounds in Todd Hewittbroth or in CASO broth (casein, soybean) indicates the MIC range forthese compounds is between 125 nM and 8 μM, the average MIC being 1.6μM. These results indicate that the compounds of the present inventionhave potent antibacterial effects against Streptococcus.

The invention claimed is:
 1. A compound having a sulfur-containing ring,or prodrugs or pharmaceutically acceptable salts thereof, of one ofFormulas I:

in which E is —CH₂— or is absent whereby the sulfur-containing ring isdirectly connected to phenyl; R₁, R₂, R₉, R₁₀, and R₁₁ if present areeach independently selected from the group consisting of hydrogen,methyl, ethyl, propyl, cyclopropyl, butyl, cyclobutyl, pentyl, hexyl,isopropryl, isobutyl, neopentyl, methoxy, and ethoxy; additionally, R₁and R₂ may connect to form a phenyl or benzofuran ring; additionally, R₉and R₁₀ may connect to form a phenyl or benzofuran ring; R₃ and R₈ areeach independently selected from the group consisting of hydrogen,methyl, ethyl, propyl, isopropyl, chlorine, fluorine, tert-butyl,methoxy, and ethoxy; R₄ and R₇ are each independently selected from thegroup consisting of hydrogen, chlorine, methyl ester, ethyl ester,methyl, ethyl, propyl, cyclopropyl, butyl, cyclobutyl, isopropryl,isobutyl, methoxy, and ethoxy; and R₅ and R₆ are each independentlyselected from the group consisting of hydrogen, cyclopentyl,cyclopropyl, furan, thiophene, trifluoromethyl, trifluoromethyl ether,methylthiol, formaldehyde, chlorine, fluorine, bromine, phenyl, methyl,ethyl, isopropyl, propyl, butyl, cyclobutyl, isobutyl, neopentyl,pentyl, methoxy, and ethoxy.
 2. The compound of claim 1 in which thecompound is according to Formula Ia; R₁ and R₂ are either independentlyselected from the group consisting of hydrogen and methyl, or form aphenyl ring whereby the ring system is naphthyl; R₉ and R₁₀ are eitherindependently selected from the group consisting of hydrogen and methyl,or form a phenyl ring whereby the ring system is naphthyl; R₁₁ ishydrogen; R₃ and R₈ are each independently selected from the groupconsisting of hydrogen, methyl, chlorine, fluorine, isopropyl,tert-butyl, and methoxy; R₄ and R₇ are each independently selected fromthe group consisting of hydrogen, methyl, chlorine, and ethyl ester; andR₅ and R₆ are each independently selected from the group consisting ofmethyl, ethyl, phenyl, hydrogen, chlorine, isopropyl, cyclopentyl,bromine, cyclopropyl, trifluoromethyl, trifluoromethyl ether,methylthiol, formaldehyde, furan, and thiophene.
 3. The compound ofclaim 2 in which E is absent.
 4. The compound of claim 3 in which R₁,R₂, R₄, R₇, R₉, and R₁₀ are each hydrogen.
 5. The compound of claim 4 inwhich R₃ and R₈ are each chlorine; and R₅ and R₆ are selected from thegroup consisting of hydrogen, methyl, and isopropyl.
 6. The compound ofclaim 1 in which the compound is5-(4,5-bis(4-chloro-2-methylphenyl)thiophen-2-yl)-1H-tetrazole or aprodrug or pharmaceutically acceptable salt thereof.
 7. The compound ofclaim 1 in which the compound is5-(4,5-bis(4-chloro-2-isopropylphenyl)thiophen-2-yl)-1H-tetrazole or aprodrug or pharmaceutically acceptable salt thereof.
 8. The compound ofclaim 1 in which the compound is5-(4-(3-butylphenyl)-5-(4-chlorobenzyl)thiophen-2-yl)-1H-tetrazole or aprodrug or pharmaceutically acceptable salt thereof.
 9. The compound ofclaim 1 in which the compound is5-(4,5-bis(4-chloro-2-isopropylphenyl)-3-methylthiophen-2-yl)-1H-tetrazoleor a prodrug or pharmaceutically acceptable salt thereof.
 10. Thecompound of claim 1 in which the compound is selected from the groupconsisting of5-(4,5-Bis(4-chloro-2-iso-propylphenyl)thiophen-2-yl)-1H-tetrazole(DNM0548),5-(4,5-Bis(2-(thiophen-3-yl)phenyl)thiophen-2-yl)-1H-tetrazole(DNM0559),5-(4-(4-Chlorophenyl)-5-(4-methylnaphthalen-1-yl)thiophen-2-yl)-1H-tetrazole(DNM0572),5-(5-(4-(5-(4-Chlorobenzyl)-4-(4-fluoronaphthalen-1-yl)thiophen-2-yl)-1H-tetrazole(DNM0587),5-(4-(Biphenyl-2-yl)-5-(4-chlorobenzyl)thiophen-2-yl)-1H-tetrazole(DNM0588),5-(4,5-bis(4-chloro-2-methylphenyl)-3-methylthiophen-2-yl)-1H-tetrazole(DNM0614), 5-(4,5-Bis(3-butylphenyl)-3-methylthiophen-2-yl)-1H-tetrazole(DNM0627), and5-(4,5-Bis(4-chloro-2-isopropylphenyl)-3-methylthiophen-2-yl)-1H-tetrazole(DNM0631), prodrugs and pharmaceutically acceptable salts thereof.
 11. Amethod of treatment of a microbial infection comprising administering aneffective amount of an antimicrobial compound of claim 1 to a patient inneed thereof.
 12. The method of treatment of claim 11 in which themicrobial infection is substantially caused by Gram-positive bacteria.13. The method of treatment of claim 12 in which the microbial infectioncomprises Staphylococcal infection.
 14. A method of treatment of amicrobial infection comprising administering an effective amount of anantimicrobial adjuvant compound of claim 1 and an effective amount of anantimicrobial compound to a patient in need thereof.
 15. The method oftreatment of claim 14 in which the microbial infection is substantiallycaused by Gram-negative bacteria.
 16. The method of treatment of claim14 in which the microbial infection is substantially caused byGram-positive bacteria.
 17. The method of treatment of claim 14 in whichthe microbial infection is polymicrobial.
 18. The method of treatment ofclaim 14 in which the microbial infection is substantially caused byPseudomonas aeruginosa.
 19. A pharmaceutical composition comprising acompound of claim 1 and a pharmaceutically acceptable excipient.